Polarization of immune cells in Theiler‘s murine ...
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Polarization ofimmune cells inTheiler‘s murineencephalomyelitis
Cut Dahlia Iskandar
Hannover 2014
Department of Pathology. Centre for Systems Neuroscience.University of Veterinary Medicine.
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1. Auflage 2014
© 2014 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH,
Gießen
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ISBN 978-3-86345-232-2
Verlag: DVG Service GmbH
Friedrichstraße 17
35392 Gießen
0641/24466
www.dvg.de
University of Veterinary Medicine Hannover Department of Pathology
Centre for Systems Neuroscience Hannover
Polarization of immune cells in Theiler’s murine encephalomyelitis
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
Awarded by the University of Veterinary Medicine Hannover
by
Cut Dahlia Iskandar
Banda Aceh
Hannover 2014
Supervisor: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ Supervision Group: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ
Prof. Dr. Martin Stangel Prof. Dr. Andrea Tipold
1st Evaluation: Prof. Dr. Wolfgang Baumgärtner, PhD/Ohio State Univ Department of Pathology
University of Veterinary Medicine Hannover Prof. Dr. Martin Stangel Clinical Neuroimmunology and Neurochemistry Department of Neurology Hannover Medical School Hannover Prof. Dr. Andrea Tipold Department of Small Animal Medicine and Surgery University of Veterinary Medicine Hannover
2nd Evaluation: Prof. Dr. C. Jane R. Welsh Department of Veterinary Integrative Biosciences and Veterinary Pathobiology Texas A&M University Date of final exam: October 10th, 2014 Parts of the thesis have been submitted to Brain Pathology and have been published in Journal of Neuroinflammation 2014, DOI 10.1186/s12974-014-0180-9
Cut Dahlia Iskandar has received financial support by the DAAD/ACEH Scholarship of Excellence. This work was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG; FOR 1103, BA 815/10-2, BE 4200/1-2 and UL 421/1-2).
PUBLICATIONS AND PRESENTATIONS
Part of the thesis have already been submitted and presented:
Publications
HERDER, V., ISKANDAR, C.D., HANSMANN, F., ELMARABET , S.A., KHAN, M.A., KALKUHL, A., DESCHL, U., BAUMGÄRTNER, W., ULRICH, R ., BEINEKE, A (2014): Dynamics changes of microglia/macrophage M1 and M2 polarization in Theiler’s murine encephalomyelitis. Submitted to Brain Pathology on 07.07.2014.
PRAJEETH, C.K., BEINEKE, A., ISKANDAR, C.D., GUDI, V., HERDER, V., GERHAUSER, I., HAIST, V., TEICH, R., HUEHN, J., BAUMGÄRTNER, W ., STANGEL, M (2014): Limited role of regulatory T cells during acute Theiler’s virus induced encephalitis in resistant C57BL/6 mice. J. Neuroinflammation; DOI 10.1186/s12974-014-0180-9
Poster presentations
ISKANDAR, C.D., V. HERDER, R. ULRICH, A. KALKHUL, U . DESCHL, F. HANSMANN, W. BAUMGÄRTNER, A. BEINEKE (2014): M1- und M2-Polarisierung von Mikroglia/Makrophagen bei der murinen Theilervirus-Enzephalomyeltis. „57. Jahrestagung und 19. Schnittseminar der Fachgruppe Pathologie der Deutschen Veterinärmedizinischen Gesellschaft (DVG)“, Fulda, Germany, March 7 – 9, 2014.
ISKANDAR, C.D., V. HERDER, W. BAUMGÄRTNER, A. BEINE KE (2014): The role of M1 and M2 Microglia and Macrophages in Theiler’s Murine Encephalomyelitis. “2nd International Workshop of Veterinary Neuroscience”, Hannover, Germany, March 20 -22, 2014.
ISKANDAR, C.D., V. HERDER, R. ULRICH, A. KALKHUL, U . DESCHL, F. HANSMANN, W. BAUMGÄRTNER, A. BEINEKE (2014): Polarization of Macrophages/Microglia in Theiler’s Murine Encephalomyelitis. “2nd Annual European Veterinary Pathology Congress”, Berlin, Germany, August 27 – 30 2014.
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Table of contents
1. Introduction ........................................................................................... …………………1 1.1. Multiple sclerosis .................................................................................. …………………1 1.1.1. Theiler’s murine encephalomyelitis ................................................... ………………....5 1.1.2. Experimental autoimmune encephalomyelitis ................................... …………………8 1.2. Microglia ............................................................................................... …………………9 1.2.1. General aspects of microglia .............................................................. …………………9 1.2.2. The role of microglia in immunology ................................................ ………………..10 1.2.3. Identification of microglia .................................................................. ………………..11 1.2.4. Phenotypes of microglia ..................................................................... ………………..12 1.2.4.1. M1 phenotype .................................................................................. ………………..12 1.2.4.2. M2 phenotype .................................................................................. ………………..13 1.3. Therapeutic strategies involving macrophage/microglia polarization .. ………………..13 1.4. Aims ...................................................................................................... ………………..16 2. Dynamic changes of microglia/macrophage M1 and M2 Polarization in Theiler’s
murine encephalomyelitis ..................................................................... ………………..17 2.1. Abstract ................................................................................................. ………………..18 2.2. Introduction ........................................................................................... ………………..19 2.3. Materials and methods .......................................................................... ………………..20 2.4. Results ................................................................................................... ………………..25 2.5. Discussion ............................................................................................. ………………..34 2.6. Acknowledgements ............................................................................... ………………..38 2.7. References ............................................................................................. ………………..39 3. Limited role of regulatory T cells during acute Theiler Virus induced encephalitis in
resistant C57BL/6 .................................................................................. ………………..45 4. General discussion .................................................................................. ………………..69 4.1. Disease phase-specific changes of macrophages/microglia polarization in Theiler’s
murine encephalomyelitis ..................................................................... ………………..69 4.2. Effects of macrophages/microglia polarization upon regeneration in the central nervous
system .................................................................................................... ………………..72 4.3. Interaction between regulatory T cells and other immune cells of the central nervous
system .................................................................................................... ………………..74 5. Conclusions ............................................................................................. ………………..78 6. Summary ................................................................................................. ………………..79 7. Zusammenfasung ................................................................................... ………………..83 8. References ............................................................................................... ………………..87 9. Attachments ............................................................................................ ………………..99 9.1. Supplemental material to chapter 2 ....................................................... ………………..99 10. Acknowledgements ............................................................................... ………………111
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List of abbreviation ALS amyotrophic lateral sclerosis APCs antigen presenting cells ATP adenosine triphosphate BBB blood brain barrier BDNF brain-derived neurotrophic factor CDV canine distemper virus CNS central nervous system CTLA-4 Cytotoxic T-lymphocyte antigen 4 DA Daniels d.p.i days post infection EAE experimental autoimmune encephalomyelitis EGF epidermal growth factor FGF-2 fibroblast growth factor 2 GDNF glial cell-derived neurotrophic factor GLUT5 glucose transporter 5 GM-CSF granulocyte macrophage colony-stimulating factor h.p.i hours post infection ICAM intercellular adhesion molecule IDO indoleamine 2,3 dioxgenase IFN interferon IFN-β interferon beta IFN-γ interferon gamma IGF-1 insulin-like growth factor 1 IL interleukin ILB4 isolectin B4 LPS lipopolysaccharide MBP myelin basic protein MHC major histocompatibility complex MOG myelin oligodendrocyte glycoprotein MPP myelin Proteolipid protein MS multiple sclerosis MRP14 myeloid-related protein 14 NGF nerve growth factor NO nitric oxide NT-3 neurotrophic factor-3 PAMPS pathogen-associated molecular patterns PDGF platelet-derived growth factor PPARs peroxisome proliferator-activated receptors PPAR γ peroxisome proliferator-activated receptors gamma PRRs pattern recognition receptors RNA ribonucleic acid
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SJL Swiss Jim Lambert TGF-β transforming growth factor beta Th T helper TLRs Toll like receptors TME Theiler’s murine encephalomyelitis TMEV Theiler’s murine encephalomyelitis virus TNF-α tumor necrosis factor alpha TO Theiler Original Tr1 T regulatory-1 Treg T regulatory
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List of figures Figure 1. Clinical course of multiple sclerosis .............................................. …………………2 Figure 2. Pathology of multiple sclerosis ...................................................... …………………3 Figure 3. Animal models for multiple sclerosis ............................................ …………………5
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1. INTRODUCTION
1.1 Multiple sclerosis
Multiple sclerosis (MS) is an autoimmunity disorder of the human central nervous system (CNS), characterized by inflammation, demyelination and axonal damage. The etiology of MS is unknown, however, a variety of viruses such as herpesviruses, retroviruses, paramyxoviruses and coronaviruses have been discussed as potential disease initiators (Mecha et al., 2013). In addition, nonspecific factors such as sex, age, latitude and genetic factors have been demonstrated to influence the development of MS. For instance, MS develops predominately in young adults and females are more often affected than males (Kurtzke, 1993; Rosati, 2001). Patients develop progressive motor and cognitive impairments with ataxia, spasticity and walking abnormalities. In addition, depression, migraine and tremors as well as bladder, intestinal and erectile dysfunction may develop. These symptoms can disappear depending on the MS form, but permanent neurological problems and disease progression may develop (Compston and Coles, 2008; Pinkston et al., 2007; Thompson et al., 2010).
Figure 1. Clinical courses of multiple sclerosis (MS). Based upon the clinical course, MS is classified into four stages: (1) relapsing-remitting (2) primary progressive (3) secondary progressive (4) progressive-relapsing MS (Minagar and Zivadinov, 2011). Most patients develop relapsing-remitting MS. Furthermore, most individuals will develop a secondary progressive disease course. The primary progressive from (10-15% of MS cases) is characterized by continuous progression of clinical symptoms beginning from the disease onset without phases of recovery (Ebers, 2005).
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Demyelination in the brain and spinal cord of MS patients is triggered by CD4+ T cells and antigen presenting cells (APCs). Myelin loss, disruption of the blood brain barrier (BBB) and axonopathies contribute to neurological disability in MS patients. Besides leukocyte infiltration, MS lesions are characterized by myelin sheath damage, oligodendrocyte loss, axonal swelling and axonal destruction as well as gliosis (Amor et al., 2010; Barnett and Prineas, 2004; Lassmann et al., 1994). During this process myelin-specific autoimmune responses can be measured. Probably APCs such as microglia and monocyte-derived macrophages are important for the initiation of immune responses and recruitment of encephalitogenic T cells. In addition to phagocytosis of myelin (myelinophagia), these cells are supposed to contribute to extensive myelin damage and oligodendrocyte dysfunction (Hendriks et al., 2005). Tissue damage by activated microglia and infiltrating macrophages is induced by various inflammatory mediators, including cytokines, chemokines, nitric oxide and reactive oxygen species (Hendriks et al., 2005; van Horssen et al., 2011). However, in addition to detrimental effects, microglia and macrophages have been demonstrated to induce remyelination and neuronal regeneration (Gay et al., 1997; Lassmann and van Horssen, 2011; Nataf, 2009). The pathology of MS is summarized in figure 2.
Figure 2. Pathology of multiple sclerosis (MS). Microglia as well as T and B cells contribute to oligodendrocyte damage and autoimmune demyelination, respectively (Lucchinetti et al., 2000; Mecha et al., 2013). Based on histology and pathogenesis, four different patterns of MS lesions can be discriminated. In pattern 1 lesions T cells and macrophages are associated with a breakdown of the blood brain barrier and demyelination. A degeneration and loss of myelin sheaths induced by antibodies and complement factors can be seen in pattern 2 lesions. Pattern 3 lesions are characterized by an infiltration of T lymphocytes, macrophage/microglia activation and distal oligodendrogliopathy. Destruction of myelin and oligodendrocyte death is indicative of pattern 4 lesions (Lassmann et al., 2001; Lucchinetti et al., 2000).
Introduction
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Early active lesions can be identified by the presence of macrophages containing myelin proteins and lipids as well as by the infiltration of macrophages expressing myeloid-related protein 14 (MRP14). Late active lesions are characterized by macrophages containing myelin debris that stains with luxol fast blue and myelin basic protein- and proteolipid protein-specific immunohistochemistry, whereas myelin oligodendrocyte glycoprotein-immunoreactivity is negative. Subsequently 27E10-positive macrophages infiltrate into lesions. Inactive lesions contain PAS-positive and MRP14-negative macrophages. Early remyelinating lesions are characterized by numerous lymphocytes and macrophages associated with clusters of thinly myelinated axons, while late remyelinating lesions (shadow plaques) consist of less macrophages, astrogliosis and numerous thinly myelinated axons (van der Valk and De Groot, 2000). Several animal models have been established to investigate different aspects of myelin disorders. As shown in figure 3, animal models of MS can be divided into four groups. Canine distemper of dogs and Visna of ruminants are naturally occurring, virus induced demyelinating diseases (Beineke et al., 2009), while Semliki forest virus infection (Fazakerley and Walker, 2003) and Theiler’s’ murine encephalomyelitis (TME) (Oleszak et al., 2004) represent experimental infectious models for MS. Experimental autoimmune encephalomyelitis (EAE) represents an autoimmune model for MS (Dal Canto et al., 1995). Furthermore, demyelination can be induced by intracerebral injection of galactocerebroside antibody and complement or by Bacillus Calmette-Guerin-induced delayed-type hypersensitivity reaction. Feeding of cuprizone induces myelin loss in the murine CNS and enables the investigation of mechanisms involved in de- and remyelination. Genetic aspects of myelin disorders can be investigated in the rumpshaker and jimpy mouse model (myelin proteolipid protein mutation) as well as in the shiverer mouse model (Baumann and Pham-Dinh, 2001). Several animal models have been established to investigate different aspects of MS (figure 3).
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Figure 3. Animal models for multiple sclerosis (MS). There are four main groups of animal models in MS research. Virus-induced demyelination can be investigated in infectious MS models, such as TME and canine distemper (Mecha et al., 2013). EAE is used to determine autoimmune aspects of demyelinating diseases (Constantinescu et al., 2011; Mecha et al., 2013). The main purpose of toxic disease models, such as the cuprizone model is to examine de- and remyelination processes of the CNS (Herder et al., 2012b). Shiverer mice and myelin associated glycoprotein deficient mice are used to investigate genetic influences in demyelinating disorders.
1.1.1 Theiler’s murine encephalomyelitis TME is a widely used viral animal model of MS. Theiler´s murine encephalomyelitis virus (TMEV) has been identified by Max Theiler in 1937 (Oleszak et al., 2004). It is a single stranded ribonucleic acid (RNA) virus belonging to the Picornaviridae family and Cardiovirus genus (Mecha et al., 2013; Oleszak et al., 2004). TMEV is divided into two subgroups: GDVII subgroup (GDVII and FA strains) and Theiler Original (TO) subgroup (Daniels (DA) and BeAn strains). The first group induces a monophasic disease, whereas the second group – as a consequence of low neurovirulence - causes a biphasic disease process with an early acute disease and late chronic demyelinating disease (Oleszak et al., 2004). Intracerebral injection of the BeAn strain causes demyelinating leukomyelitis with virus persistence in glial cells in susceptible mice strains, such as Swiss Jim Lambert (SJL) mice (Kummerfeld et al., 2009; Zoecklein et al., 2003). During the early phase of the infection a polioencephalitis with primary infection of neurons in the cortex and hippocampus can be observed. In general, the onset of myelin loss depends on the viral dose and the age of animals. At this, approximately 35 to 45 days post infection (dpi) progressive neurological
Introduction
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deficits, such as waddling gait and hind leg paralyses due to spinal cord demyelination can be observed in TMEV-infected mice (Mecha et al., 2013; Oleszak et al., 2004). Similar to MS, TMEV induced CNS lesions are characterized by demyelination and axonal damage (Mecha et al., 2013). TMEV triggers innate immune responses, followed by adaptive immune responses. However, despite the occurrence of virus specific humoral and cellular immune responses, viral elimination is insufficient, which causes viral persistence in glial cells (Kim et al., 2005b). Referring to this, a key event in the pathogenesis is the stimulation of delayed-type hypersensitivity reaction and probably myelin-specific autoimmunity by prolonged viral epitope presentation (Gerhauser et al., 2012; Liuzzi et al., 1995a; Roussarie et al., 2007). In addition to T cell-mediated immunopathologies, macrophages and microglial cells contribute to myelin damage by the release of myelinotoxic factors (bystander demyelination). Moreover, activated glial cells, including microglia, enhance immune mediated tissue damage by the production of pro-inflammatory cytokines and chemokines which causes an increased CNS-infiltration of lymphocytes (Oleszak et al., 2004). Resident microglial cells play an important role for antigen presentation at disease onset (Kennedy et al., 1998), which leads to an activation of CD4+ and CD8+ T cells in the late chronic demyelinating disease phase (Miller, 1997). TMEV persists in macrophages and glial cells such as microglia of SJL/J mice (Clatch et al., 1987; Lipton and Melvold, 1984; Lipton et al., 1995). This process is associated with the up-regulation of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-12, IL-18, and type I interferon (IFN) as well as major histocompatibility complex II (MHC II) and co-stimulatory molecules (B7-1, B7-2 and CD 40) (Dale et al., 2008; Mackaness, 1977; O'Shea et al., 2008; Olson et al., 2001). Recent experiments revealed a phenotype switch of TMEV-infected microglia in vitro with high IL-10 and low IL-12 mRNA levels at 48 hours post infection (hpi) and low IL-10 and high IL-12 as well as TNF mRNA levels at 240 hpi (Gerhauser et al., 2012). In vivo studies described an up-regulation of IL-1, IL-12 and IL-10 in the early phase of TME (at 168 hpi), while strong IL-12 gene expression was found in the CNS of susceptible mice during the late demyelinating phase of TME, indicative of T helper 1 (Th1) immune responses (Sato et al., 1997). In addition, the up-regulation of IL-12 may facilitate a switch of the microglia phenotype from an anti-inflammatory (alternative activated M2) type to a pro-inflammatory M1 phenotype (Bright et al., 1999; Palma and Kim, 2004). M1 microglial responses are supposed to cause antiviral effects but probably also immune mediated tissue damage in TMEV-infected mice (Gerhauser et al., 2012; Kim et al., 2001; Mantovani et al., 2004). However, besides their detrimental functions, macrophage/microglia phagocytose myelin debris, which is an important prerequisite for neuroregeneration and remyelination, respectively. Thus, microglia and macrophages are supposed to have both pro-inflammatory and anti-inflammatory properties in TMEV-infected mice, as discussed for degenerative CNS
Introduction
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disorders (Kigerl et al., 2009). However, so far, the potential dual role of microglia/macrophages in the pathogenesis of TME, especially the M1 and M2 phenotype polarization of these cells has not been investigated in detail. 1.1.2 Experimental autoimmune encephalomyelitis
EAE is commonly used for the investigation of autoimmune aspects of MS and the development of immunomodulatory therapies of human inflammatory demyelinating diseases (Andersson and Karlsson, 2004; Constantinescu et al., 2011; Steffen et al., 1994; t Hart et al., 2011) Similar to MS, an inflammatory demyelinating disease of the CNS can be observed in EAE (Constantinescu et al., 2011; Steffen et al., 1994). In the early 1930s, for the first time, Thomas M. Rivers and his colleagues induced EAE in guinea pigs and rats. Nowadays, protocols to induce EAE in a variety of species including mice, rabbits, goats, hamsters, dogs, sheep, marmots and chickens have been established (Baxter, 2007; Kuerten et al., 2007). Similar to TME, EAE in mice is influenced by the genetic background of the animals. For instance, SJL mice represent a susceptible strain which develops autoimmune demyelination (Steffen et al., 1994). EAE in susceptible animals can be induced by the adoptive transfer of myelin-specific lymphocytes or by the immunization with CNS antigens, including myelin basic protein (MBP), myelin proteolipid protein (MPP) and myelin oligodendrocyte glycoprotein (MOG). Following the immunization, antigen-specific T cells are activated in peripheral lymphoid organs. Reactivation of these primed T cells occurs after CNS infiltration by local APCs such as microglia, which causes immune mediated tissue damage (Andersson and Karlsson, 2004). The main effector cells in EAE are interferon gamma (IFN-γ) producing Th1 and Th17. Both cell types are activated in peripheral lymphoid organs by dendritic cells. After crossing the BBB, these T cells are reactivated in the CNS by antigen-presenting cells. At this, microglia present antigen in conjunction with MHC II to CD4+ Th cells. Subsequent production of inflammatory cytokines and toxic factors contribute to myelin and axon damage. This process is enhanced by microglia by the release of factors that attract further inflammatory cells (Constantinescu et al., 2011). On the other hand, activated T cells secrete cytokines such as IFN-γ which further contributes to the activation of microglia. Microglial products including TNF-α and nitric oxide (NO) damage oligodendrocytes. Furthermore, IL-1, IL-6 and TNF-α produced by microglia induce astrogliosis and regulate expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecute 1 and E-selectin on astrocytes (Merrill et al., 1993; Zajicek et al., 1992).
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1.2 Microglia
1.2.1 General aspects of microglia
Microglial cells represent the main APC of the CNS. They are supposed to derive from bone marrow macrophages. Pio Del Rio-Hortega described microglial cells in 1932 and divided them into 3 forms: ramified microglia, phagocytic microglia and amoeboid microglia (Kettenmann et al., 2011). Microglia share many morphological and functional similarities with parenchymal tissue macrophages. An important function of microglia is to maintain immune homeostasis of the CNS (Saijo and Glass, 2011). They have the ability to phagocytize debris and to produce several cytokines and chemokines to initiate tissue repair following injury and to induce innate and adaptive immune responses, respectively (Aguzzi et al., 2013; Black and Waxman, 2012; Ensinger et al., 2010; Saijo and Glass, 2011). Microglia make up 10% of glial cells in the CNS (Aguzzi et al., 2013). Microglia get activated by a variety of signals induced by cellular stress, cell damage and T cell released cytokines (inside factor) as well as by pathogens-associated molecules (outside factor) (Aloisi, 2001). Accordingly, microglial activation can be achieved by ligation of Toll like receptors (TLRs) and proinflammatory cytokines such as TNFα and IL-6. Subsequent increased expression of MHC class II and costimulatory molecules (CD80, CD86, CD40) on microglia are required to present antigens to CD4+ T cells (Olson, 2010). Similar functions for the initiation of innate and adaptive immune responses can be observed for CNS-infiltrating macrophages (Martinez et al., 2008). However, it has been observed that microglia have a higher phagocytic capacity than infiltrating macrophages, despite similar morphology (Durafourt et al., 2012). 1.2.2 The role of microglia in immunology Microglia play a pivotal role for immune homeostasis in the CNS and protection against infectious agents and neurodegeneration. They are activated during injury and infectious disease via a variety of receptors, such as immunoglobulin superfamily, complement receptors, cytokine/chemokine receptors, Toll-like receptors, CD14 receptors, mannose receptors, purinogenic receptors, opioid receptors, cannabinoid receptors and benzodiazepine receptors. Secretory products of microglia include cytokines, chemokines, matrix metalloproteinase, free radicals, eicosanoids, growth factors, proteases, cathepsins, quinolinic acid, amyloid precursor protein and complement factors (Rock et al., 2004). Microglia are the main source of inflammatory mediators and have the ability to process and present antigen to T cells (Aloisi, 2001). Therefore, they play a central role in innate and
Introduction
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adaptive immunity. Moreover, microglia collaborate with other glial cells such as oligodendrocytes and astrocytes to induce inflammatory responses (Gao and Tsirka, 2011). Microglia of the brain and spinal cord exhibit different functions (e.g. phagocytic capacity and reactive oxygen species generation; Ensinger et al., 2010). In addition, differences can also be observed between microglia in the ventral horn and dorsal horn of the spinal cord (Olson, 2010). Therefore, topographical variations in the functionality of microglia have to be considered in the pathogenesis of CNS disorders. 1.2.3 Identification of microglia Visualization of microglia can be achieved by labeling cell surface-associated or intracellular molecules in brain tissue sections, organotypic slice cultures or mixed brain cell cultures. Similar to endothelial cells, microglia express glycan moieties which can be identified by Griffonia simplicifolia isolectin B4 (ILB4) or tomato lectin (Boya et al., 1991; Streit and Kreutzberg, 1987) which enable to distinguish microglia cells from other brain cells, but not from CNS-infiltrating macrophages (Thomas, 1999). Molecules targeted by immunological techniques include CD11b/CD 18, complement receptor 3 and MAC 1 (Ma et al., 2003), immunoglobulin receptors (CD16/32/64, FcγRIII/II/I), CD45 (leukocyte common antigen), CD68 (macrosialin), CD163 (scavenger receptor M130, ED2), CD169 (sialoadhesion, siglec-1), CD204 (MSR), F4/80 antigen, β-glucan receptor dectin-1, and mannose receptor (CD206). CD11b and ionized calcium-binding adapter molecule 1 (Iba1) increase upon microglia activation (Ito et al., 1998). Difficulties arise under pathological conditions, since activated microglia and infiltrating macrophages show an overlap of marker expression (Zhang et al., 2002): a. CD45 low: parenchymal microglia. b. CD45 intermediate: other CNS-associated macrophages. At least in human brain tissue the glucose transporter 5 (GLUT5) is restricted to microglia and serves as a marker for resting and activated cells (Horikoshi et al., 2003; Maher et al., 1994; Vannucci et al., 1997). 1.2.4 Phenotypes of microglia There are two major subgroups of microglia which can polarize into the M1 phenotype (classically activated) and M2 phenotype (alternatively activated). Basically, M1 microglial cells are characterized by the ability to induce pro-inflammatory cytokines, while M2 cells produce predominately anti-inflammatory and immunomodulatory cytokines. During infectious diseases of the CNS, M1 microglia promote inflammation, which leads to pathogen-specific protective immune responses but also to immune mediated tissue damage. The M1 polarization of microglia is observed predominately during the acute infection phase. In contrast, M2 cells promote tissue repair during advanced disease stages. However, due to their immunosuppressive properties which dampen the protective immunity excessive M2 microglial responses could favor persistent infection.
Introduction
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1.2.4.1 M1 phenotype M1 polarization is achieved by activating cytokines such IFN-γ, TNFα, and granulocyte macrophage colony-stimulating factor (GM-CSF) (Durafourt et al., 2012; Laskin, 2009). In addition, microglia express pattern recognition receptors (PRRs), which bind to pathogen-associated molecular patterns (PAMPS), including lipopolysaccharide (LPS). Following activation, microglia secrete TNF and IL-1β as well as chemokines (e.g. CC-chemokine ligand 2), reactive oxygen species and NO. Moreover, M1 microglia express MHC class II which is pivotal for antigen presentation and T cell activation, respectively. Additionally, M1 microglia produce IL-12, promoting Th1 immune responses in the CNS (Saijo and Glass, 2011). 1.2.4.2 M2 phenotype
M2 polarized microglia are able to reduce inflammation and stimulate tissue regeneration. The peroxisome proliferator-activated receptors (PPARs) of macrophages or the peroxisome proliferator-activated receptors gamma (PPARγ) of microglia can induce and regulate M2 phenotype expression in mice (Aguzzi et al., 2013). The M2 phenotype is divided in 3 subgroups: M2a, M2b and M2c (Laskin, 2009). M2a cells play an important role for phagocytosis and tissue repair. IL-4 and IL-13 represent activating signals for this subgroup. Immune complexes and IL-1ß represent activating signals for M2b cells, IL-10, transforming growth factor beta (TGFβ) and glucocorticoids for M2c cells. The main functions of M2c microglia are down-regulation of M1 responses and polarization of M1 cells into M2 cells, respectively. They also promote wound healing and tissue remodeling (Andjelkovic et al., 1998; Durafourt et al., 2012; Laskin, 2009; Vereyken et al., 2011). In general, M2 cells inhibit immune mediated tissue damage and favor matrix remodeling and angiogenesis. Moreover, M2 cells can stimulate the activation of regulatory T cells (Treg). During TMEV infection, Th2 immune responses are induced by M2 microglia (Gordon and Martinez, 2010). The phagocytic activity of M2 cells is induced by FCγR1A (CD64) (Durafourt et al., 2012). The most striking feature of M2 microglia is their ability to enhance CNS repair (Aguzzi et al., 2013). 1.3 Therapeutic strategies involving macrophage/microglia polarization Modulating the microglia polarization of the spinal cord might represent a prerequisite to stimulate endogenous regeneration and future transplantation approaches (Kobayashi et al., 2013). Dimethyl fumarate (Tecfidera) is a methyl ester of fumaric acid which has been shown to reduce relapse rate in MS patients in clinical trials. Dimethyl fumarate is supposed to have immunomodulatory properties without immunosuppressive effects, e.g. by decreasing the expression of nitric oxide, IL-1β, IL-6 and TNF-α in microglia. It is also able to reduce the
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infiltration of macrophages in the brain in murine EAE (Schilling et al., 2006; Wierinckx et al., 2005). Moreover, the drug fasudil hydrochloride (Fasudil), a Rho-kinase inhibitor, delays the disease onset and ameliorates the clinical severity of EAE by promoting the shift of M1- to M2-phenotype of macrophages/microglia (Liu et al., 2013). The therapeutic effect of fasudil is a consequence of reduced expression of IL-1β, IL-6, and TNF-α and enhanced IL-10 production associated with an increased expression of arginase-1 as observed in spinal cord macrophages/microglia of treated mice (Hou et al., 2012). Similarly, the phenyl aziridine precursor Compound A, a plant-derived ligand of glucocorticoid receptors increases the yield of anti-inflammatory M2-type macrophages in vitro. It also inhibits the progression of neuropathic pain and represses spinal cord microglia in rat experimental autoimmune neuritis (Zhang et al., 2009). Glatiramer acetate (Copaxone) is an immunomodulatory drug that was designed to mimic myelin basic protein (Johnson et al., 1995). The substance exerts therapeutic effects in relapsing remitting MS, probably mediated by the induction of Th2 responses and the production of anti-inflammatory cytokines, such as IL-10 and TGF-β. In addition, glatiramer acetate has been suggested to promote M2-polarization of macrophages/microglia with reduced NO and TNF-α release (Iarlori et al., 2008; Jung et al., 2004; Weber et al., 2004). In addition the drug is able to increase the phagocytic activity of microglia and clearance of myelin debris which supports remyelination (Rawji and Yong, 2013; Trojano et al., 2003). The drug interferon-β (IFN-β) (Avonex, Betaferon) is used to reduce the rate and severity of MS relapses. IFN-β has been shown to diminish the antigen presenting capacity (reduced MHC class II expression) and respiratory burst of macrophages/microglia that potentially leads to decreased responses of encephalitogenic T cells and reduced brain damage, respectively (Hall et al., 1997; Rawji and Yong, 2013). Besides preventing leukocyte recruitment to the CNS in MS patients, the application of Fingolimod (Gilenya) induces an anti-inflammatory phenotype of macrophages associated with a reduced production of free radicals (Hughes et al., 2008; Rosen and Goetzl, 2005). The intercalating substance Mitoxantrone (Novatrone) is used to treat cancer. In addition, its immunosuppressive effect results in reduced migration and activation of monocytes in the brain and reduced macrophage-mediated degradation of myelin in MS patients (Fox, 2004; Kopadze et al., 2006; Vollmer et al., 2010; Watson et al., 1991). The tetracycline-class antibiotic minocycline (Minocin) selectively inhibits M1 polarization of microglia which delays the onset and mortality in mouse models for amyotrophic lateral sclerosis (ALS) (Kobayashi et al., 2013). Furthermore, minocycline reduces inflammatory demyelination in EAE. However, remyelination following ethidium bromide-induced demyelination in rats is impaired when macrophage activation is reduced by minocycline administration, demonstrating the pivotal and complex role of macrophages/microglia for neuroregeneration (Li et al., 2005; Miron and Franklin, 2014). 1.4 Aims Microglia and CNS-infiltrating macrophages represent target cells for viral persistence in TME and contribute to myelin damage by delayed-type hypersensitivity, bystander
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demyelination and the induction of myelin-specific autoimmunity. Similarly, M1-polarized cells foster immunopathology in primary autoimmune myelin loss disorders, such as EAE. Moreover, an imbalance between pro-inflammatory M1- and neuroprotective M2-type cells is supposed to contribute to excessive inflammation in traumatic CNS diseases (Kigerl et al., 2009) and selective inhibition of M1-type microglia reduces neurodegeneration in mouse models for amyotrophic lateral sclerosis (Kobayashi et al. 2013). Recent studies have demonstrated also that the switch of M1- into M2-type macrophages/microglia is required for efficient oligodendrocyte differentiation and myelin repair following toxin-induced demyelination in rodents (Miron et al., 2013). However, similar to regulatory T cells, M2-type macrophages/microglia have the potential to dampen protective immune responses which leads to disease exacerbation or persistent inflammation in infectious MS models. So far, polarization of macrophages/microglia in TME has not yet been investigated in detail. Thus, the aim of the present study was to determine dynamic changes of different microglia/macrophage populations in the spinal cord of TMEV-infected mice and to provide a comprehensive database of M1/M2-related genes involved in the initiation and progression of virus-induced demyelination. In order to get further insights into the role of immune homeostasis for disease resistance and antiviral immunity in TME, the effect of selective ablation of Foxp3+ regulatory T cells upon neuroinflammation, including microglial responses, was investigated.
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2. Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s
murine encephalomyelitis
Vanessa Herder1,2*, Cut Dahlia Iskandar1,2*, Florian Hansmann1,2, Suliman Ahmed Elmarabet1, Muhammad Akram Khan1,2, Arno Kalkuhl3, Ulrich Deschl3, Wolfgang
Baumgärtner1,2, Reiner Ulrich1,2,♦, Andreas Beineke1,2,4,♦
1Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany 2Center for Systems Neuroscience, Hannover, Germany 3Department of Non-Clinical Drug Safety, Boehringer Ingelheim Pharma GmbH & Co. KG,
Biberach (Riss), Germany Contribution of the PhD student: C.D. Iskandar contributed to the study design and writing of the manuscript and performed immunofluorescence microscopy, analyses of M1- and M2- related gene expression data, and statistical analyses.
*authors have contributed equally; ♦authors have contributed equally
4corresponding author Prof. Dr. Andreas Beineke, Dipl. ECVP Department of Pathology University of Veterinary Medicine Hannover Bünteweg 17 D-30559 Hannover, Germany Mail: [email protected] Phone: 0049-511-953-8640 Fax: 0049-511-953-8675
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2.1 Abstract
Microglia and macrophages play a central role for demyelination in Theiler’s murine encephalomyelitis (TME) virus-infection, a commonly used infectious animal model for chronic-progressive multiple sclerosis. In order to determine dynamic changes of microglia/macrophage polarization in TME, the spinal cord of SJL mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and chronic demyelinating leukomyelitis was confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase, indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TMEV-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1-polarization, while mounting M2-polarization of macrophages/microglia together with sustained prominent M1-related gene expression were present during the chronic progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32+ M1- and arginase-1+ M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.
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2.2 Introduction
Multiple sclerosis (MS), one of the most frequent central nervous system (CNS) diseases in young adults, is a chronic demyelinating disease of unknown etiology and possibly multifactorial causes (Compston and Coles, 2008). Based on the generation of myelin-specific immune responses, MS is regarded as an autoimmune disease (Bernard and de Rosbo, 1991; Ota et al., 1990), presumably triggered by virus infections (Kakalacheva et al., 2011; Munz et al., 2009). Due to clinical and pathological similarities, Theiler’s murine encephalomyelitis (TME) represents a commonly used infectious animal model for the chronic-progressive form of human MS (Dal Canto et al., 1995; Miller et al., 2001; Monteyne, 1999; Raddatz et al., 2014). Following intracerebral infection with a low virulent BeAn-strain of Theiler’s murine encephalomyelitis virus (TMEV) susceptible mouse strains develop persistent CNS infection with immune mediated spinal cord demyelination and remyelination failure (Haist et al., 2012; Hou et al., 2009; Kumnok et al., 2008; Lipton, 1975; McMahon et al., 2005; Miller et al., 1997; Tsunoda, 2008; Tsunoda and Fujinami, 1996; Ulrich et al., 2010). Microglia and CNS-infiltrating macrophages play a central role in the pathogenesis of TMEV-induced demyelination. They represent target cells for viral persistence during the chronic disease phase (Kummerfeld et al., 2012; Rossi et al., 1997) and contribute to myelin damage by the release of myelinotoxic factors (bystander demyelination), delayed-type hypersensitivity reaction and induction of myelin-specific autoimmunity (Liuzzi et al., 1995b; Mecha et al., 2013). Similarly, microglia induces myelin damage also in autoimmune and toxic rodent models for MS, such as experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination, respectively (Liu et al., 2013; Skripuletz et al., 2010; Voss et al., 2012). The current concept of microglia/macrophages plasticity describes different cell populations with distinct and even opposing functions. For instance, M1-type microglia/macrophages promote inflammation, which leads to protective immunity against pathogens but if uncontrolled also to immune mediated tissue damage by the release of pro-inflammatory cytokines, reactive oxygen species and nitric oxide (Pinteaux-Jones et al., 2008; Prajeeth et al., 2014). In contrast, M2-type cells exhibit neuroprotective properties usually during advanced disease stages due to phagocytosis of debris, promoting tissue repair and termination of neuroinflammation by down-regulating M1- and Th1-immune responses (Laskin, 2009). So far, only a few reports mention the polarizing effects of TMEV upon microglia in vitro (Gerhauser et al., 2012). Moreover, M1- and M2-type cells represent merely two extremes of the polarization continuum and macrophages/microglia with an intermediate activation status can be observed inter alia in demyelinating MS lesions (75), demonstrating the need for quantitative analyses of M1/M2-related factors in myelin disorders. Thus, the aim of the present study was to determine dynamic changes of microglia/macrophage polarization in the
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spinal cord of SJL mice during the initiation and progression of TME with the aid of immunofluorescence and gene expression profiling. 2.3 Materials and methods
Experimental design Five-week-old female SJL/J mice (Harlan, Borchen, Germany) were inoculated into the right cerebral hemisphere with 1.63x106 plaque-forming units/mouse of the BeAn-strain of TMEV in 20µl Dulbecco’s Modified Eagle Medium (PAA Laboratories, Cölbe, Germany) with 2% fetal calf serum and 50µg/kg gentamicin. Mock-infected animals received 20µl of the vehicle only. Inoculation was carried under general anesthesia with medetomidine (0.5 mg/kg, Domitor, Pfizer, Karlsruhe, Germany) and ketamine (100 mg/kg, Ketamine 10%, WDT eG, Garbsen, Germany). All experiments were performed in groups of six TMEV- and 3-6 mock-infected mice, euthanized 14, 42, 98 and 196 days post infection (dpi). For histology, immunohistochemistry and special stains, thoracic spinal cord segments were removed immediately after death and fixed in 10% formalin for 24 hours, decalcified in disodium-ethylenediaminetetraacetate for 48 h and subsequently embedded in paraffin wax. For microarray analysis and immunofluorescence, spinal cords were immediately removed, snap-frozen in liquid nitrogen and stored at -80°C (Herder et al., 2012a; Navarrete-Talloni et al., 2010b; Ulrich et al., 2010). The animal experiments were approved and authorized by the local authorities (Niedersächsisches Landesamt für Verbraucherschutz- und Lebensmittelsicherheit [LAVES], Oldenburg, Germany, permission number: 33.9.42502-04/07/1331, 509c-42502-02/589 and 33-42502-05/963). Histology Leukomyelitis was evaluated on hematoxylin and eosin (HE)-stained transversal sections using a semiquantitative scoring system based upon the degree of perivascular infiltrates: 0 = no changes, 1 = scattered perivascular infiltrates, 2 = 2 to 3 layers of perivascular inflammatory cells, 3 = more than 3 layers of perivascular inflammatory cells, as described previously (Gerhauser et al., 2007). For the evaluation of myelin loss, serial sections of spinal cord were stained with Luxol fast blue-cresyl violet (LFB-CV) and the degree of demyelination was semi-quantitatively evaluated as follows: 0 = no change, 1 = 25%, 2 = 25-50% and 3 = 50-100% of the white matter affected (Gerhauser et al., 2007). The scoring was performed separately on all 4 quarters of spinal cord transversal sections. For each animal the arithmetic average of leukomyelitis and myelin loss was calculated. Histological data used for the present study were generated in our previous studies (Ulrich et al., 2006; Ulrich et al., 2010).
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Immunohistochemistry Immunohistochemistry was performed using a polyclonal rabbit anti-TMEV capsid protein VP1-specific antibody, as described before (Kummerfeld et al., 2009). Briefly, for blocking of the endogenous peroxidase, formalin-fixed, paraffin-embedded tissue sections were treated with 0.5% H2O2 diluted in methanol for 30 minutes at room temperature. Subsequently, slides were incubated with the primary antibody at a dilution of 1:2000 for 16 hours at 4°C. Goat-anti-rabbit IgG diluted 1:200 (BA9200, H+L, Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody for one hour at room temperature. Sections used as negative controls were incubated with rabbit normal serum at a dilution of 1:2000 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Slides were subsequently incubated with the peroxidase-conjugated avidin-biotin complex (ABC method, PK-6000, Vector laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. After the positive antigen-antibody reaction visualization by incubation with 3.3-diaminobenzidine-tetrachloride in 0.1M imidazole, sections were counterstained with Mayer’s hematoxylin. Immunofluorescence Methanol-fixed frozen sections of the thoracic spinal cord were rinsed in 0.1% Triton X-100 (Sigma-Aldrich, Taufkirchen, Germany) in phosphate buffered saline (PBS) for 30 minutes. Non-specific binding was blocked with 20% goat or horse serum, respectively, diluted in PBS/0.1% Triton X-100/1% bovine serum albumin for 30 min. After washing with 0.1 % Triton X-100 in PBS, slides were incubated with primary CD68- (monoclonal rat anti-mouse antibody, Ab53444, clone FA-11, Abcam Ltd.; dilution 1:200) and CD107b- (monoclonal rat anti-mouse antibody MCA2293, clone M3/84, AbD Serotec; dilution 1:200) for the detection of macrophages/microglia. For visualization of M1-type macrophages/microglia a CD16/32-specific antibody (monoclonal rat anti-mouse, 553141, clone 2.4G2, BD Pharmingen; dilution 1:25) and for M2-type cells an arginase-1-antibody (polyclonal goat anti-human antibody, SC-18351, Santa Cruz Biotechnology; dilution 1:50) was used. Slides were incubated for one hour, followed by washing in PBS/0.1% Triton X-100. As negative control, slides were incubated with goat or rat serum in the same concentration as the primary antibodies. Subsequently slides were incubated with secondary Cy3-conjugated goat anti-rat IgG antibody or Dylight 488-conjugated donkey anti-goat IgG antibody (Jackson ImmunoResearch Laboratories, Dianova, Hamburg, Germany), respectively, for one hour at room temperature and afterwards washed in PBS. Cell nuclei were stained using 1.0 % bisbenzimide for 10 minutes and slides were mounted with fluorescent mounting medium (Dako Diagnostika, Hamburg, Germany). Statistical analyses For non-category data obtained by histology, immunohistochemistry and immunofluorescence, a Mann-Whitney-U-test was performed. A p-value of less than 0.05 was considered as statistically significant.
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Electron microscopy Electron microscopy was performed as described previously (Kreutzer et al., 2012; Ulrich et al., 2008). Spinal cord samples were fixed with 2.5% glutaraldehyde and incubated overnight at 4°C. Post-fixation was performed in 1% aqueous osmium tetroxide and after five washes in cacodylate buffer (five minutes each) samples were dehydrated through series of graded alcohols and embedded in Epon 812 medium. Semi-thin sections were cut on a microtome (Ultracut Reichert-Jung, Leica Microsystems, Germany) and stained with uranyl citrate for 15 minutes. After eight washing steps samples were incubated with lead citrate for seven minutes. Ultra-thin sections were cut with a diamond knife (Diatome, USA) and transferred to copper grids. The affected white matter were examined by a transmission electron microscope (EM 10C, Zeiss, Germany). Microarray analyses RNA was isolated from frozen spinal cord samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany), amplified and labeled using the Message Amp II-Biotin Enhanced Kit (Ambion, Austin, USA) and hybridized to GeneChip mouse genome 430 2.0 arrays (Affymetrix, Santa Clara, USA) as described (Ulrich et al., 2010). Six biological replicates were used per group and time point, except for five TMEV-infected mice at 98 dpi. Background adjustment and quantile normalization was performed using RMAExpress (Bolstad et al., 2003). MIAME compliant data set are deposited in the ArrayExpress database (E-MEXP-1717; http://www.ebi.ac.uk/arrayexpress). Selection of M1- and M2-associated genes For molecular characterization of macrophage/microglia polarization a data set of genes differentially expressed in the spinal cord of TMEV-infected SJL mice obtained in our previous global gene expression analysis was used (Ulrich et al., 2010). The present analyses focused on a list of genes associated with M1- or M2-polarization of microglia/macrophages (Supplemental Table S1) according to peer-reviewed publications (David and Kroner, 2011; Durafourt et al., 2012; Kigerl et al., 2009; Martinez., 2006). The fold change was calculated as the ratio of the inverse-transformed arithmetic means of the log2-transformed expression values of TMEV-infected versus mock-infected mice. Down-regulations are shown as negative reciprocal values. Independent pair-wise Mann-Whitney-U-tests (IBM SPSS Statistics, version 20, IBM Corporation, Armonk, USA) comparing TMEV- and mock-infected mice were calculated followed by adaption of the p-values according to the method described by Storey and Tibshirani using QVALUE 1.0 (Storey and Tibshirani, 2003). Significantly differentially expressed genes between TMEV- and mock-infected mice were
selected employing a q-value ≤0.05 cutoff combined with a ≥2.0 or ≤-2.0 fold-change filter. The relative percentage of differentially expressed M1- versus M2-marker genes was
compared for each time point employing Fisher’s exact tests (p-value ≤0.05).
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2.4 Results
Histology and immunohistochemistry Histological examination of the HE-stained spinal cord sections revealed a mononuclear inflammation (leukomyelitis) within the white matter of TMEV-infected mice beginning at 14 dpi. The inflammatory changes increased towards 98 dpi and were significantly increased compared to mock-infected control animals at all investigated time points: 14 dpi (p=0.011), 42 dpi (p=0.002), 98 dpi (p=0.013) and 196 dpi (p=0.002; Figure 1 and 2). The amount of demyelination increased until 196 dpi (Figure 1 and 2). Figure 1. Histological lesions in the spinal cord of Theiler´s murine encephalomyelitis virus-infected mice. A) Lymphocytic meningitis (arrows) and B) mild vacuolization of the spinal cord white matter in an infected animal at 42 days post infection. C) Prominent infiltration of macrophages/microglia in the spinal cord and lymphocytic meningitis (arrow) at 196 days post infection. D) Demyelination of the spinal cord white matter (asterisks) at 196 days post infection. E) Higher magnification of C) showing activated macrophages/microglia with a foamy cytoplasm (gitter cells). F) Note accumulation of myelin debris within the cytoplasm of macrophages/microglia, indicative of myelinophagia. GM = gray matter; bars = 300µm (A-D) and 30µm (E-F); hematoxylin-eosin stain (A,C,E), luxol fast blue stain (B,D,F).
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Figure 2. Scoring of demyelinating leukomyelitis in Theiler´s murine encephalomyelitis virus-infected mice. A) Histology reveals inflammatory responses in the spinal cord (leukomyelitis) at all investigated time points. B) Detection of demyelination in the spinal cord white matter at 42, 98 and 196 days post infection. dpi = days post infection; mock = mock-infected control mice; TMEV = Theiler´s murine encephalomyelitis virus-infected mice;
∗ = significant difference (p≤0.05, Mann-Whitney-U-test). Box and whisker plots display median and quartiles with maximum and minimum values.
At 3 investigated time points (42, 98 and 196 dpi), demyelination in the spinal cord of TMEV-infected SJL-mice was significantly increased compared to mock-infected control mice (p=0.002, p=0.007, p=0.002) as determined by the myelin stain LFB-CV. Immunohistochemistry for the detection of virus protein in the spinal cord of TMEV-infected mice revealed infection at all investigated time points (14, 42, 98, and 196 dpi). Positive cells were located predominantly in the ventral aspects of the white matter. No positive signals were observed in mock-infected control mice (Supplemental figure S1).
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Figure S1. Quantification of Theiler´s murine encephalomyelitis (TMEV)-infected cells in the murine spinal
cord. dpi = days post infection; mock = mock-infected control mice; ∗ = significant difference (p≤0.05, Mann-Whitney-U-test). Box and whisker plots display median and quartiles with maximum and minimum values. Electron microscopy
Descriptive ultrastructural analyses revealed subtle myelin changes before the onset of overt demyelination at 14 dpi in an average of 0.3% of investigated axons, characterized by
vacuolization of myelin sheaths. At 42 dpi 2.2% of axons showed myelin sheath vacuolization and 5.8% of axons showed a complete loss of myelin (Figure 3). Figure 3. Ultrastructural analyses of the spinal cord white matter of Theiler`s murine encephalomyelitis virus-infected mice by transmission electron microscopy. A) Macrophages/microglia containing phagocytized myelin fragments (white asterisks) at 42 days post infection, characteristic of myelinophagia (M = nucleus of a macrophage/microglial cell; magnification 13300x). B) Demyelinated axons (black asterisks) lacking myelin sheaths and focal myelin vacuolization (arrow) in an infected mouse at 196 days post infection. For comparison, myelinated axons with intact myelin sheaths are labelled with triangles (magnification 6600x). C) Oligodendrocyte in proximity to remyelinated axons with thin myelin sheaths (black asterisks) during late chronic infection phase (196 days post infection), indicative of oligodendrocyte-mediated remyelination. Normally myelinated axons are labelled with triangles (O = nucleus of an oligodendrocyte; magnification 5300x). D) Remyelination by Schwann cells in a demyelinated area at 196 days post infection. Newly formed myelin sheaths are indicated by arrows (S = nucleus of a Schwann cell; magnification 6650x).
At 98 dpi an average of 2.8% of vacuolated myelin sheaths were observed and 8.4% of axons were totally denuded in demyelinated foci. At 196 dpi 5.0% of axons within white matter
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lesions showed a complete loss of myelin sheath, while 2.5% of axons show oligodendrocyte-type remyelination and 0.7% Schwann cell-type remyelination (Figure 2), indicative of beginning but abortive myelin repair (Ulrich et al., 2008). Phagocytosis of myelin fragments associated with denuded axons, representing a hallmark of active demyelination, was observed starting 42 dpi. At this time point an average of 40.2% of microglia/macrophages displayed gitter cell morphology with phagocytized myelin in the cytoplasm (myelinophages; Figure 3). At 98 and 196 dpi, 50.1% and 51.5% of investigated macrophages/microglia represent myelinophages. In addition, phagocytized apoptotic bodies were present in an average of 9.3% of macrophages/microglia at 42 dpi, followed by a decline at 98 (0.7%) and 198 dpi (0.5%). DNA microarray analyses In order to get insights into polarization related to microglia/macrophages, DNA microarray analyses of spinal cord tissue have been performed. A total of 151 genes related to macrophages/microglia-polarization were extracted from peer-reviewed publications, of which 72 and 66 were unequivocally assigned as M1- and M2-marker genes, respectively. Thirteen genes were assigned to both polarization types (supplemental table S1). A total of 59 genes (39.1%) were differentially expressed in TMEV-infected mice over the study period (Figure 4, supplemental table S2). Most strikingly, although the number of differentially expressed genes increased over the study period for both phenotypes, comparison of the relative proportion of differentially expressed M1- versus M2-marker genes revealed a significantly higher percentage of differentially expressed M1-marker genes at 14 (p=0.035) and 42 dpi (p = 0.016). In addition, a statistical tendency (p = 0.078) of increased M1-associated genes was observed at 98 dpi, whereas a comparable proportion of M1- and M2-marker genes was detected at later time points (Figure 4).
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Figure 4. Expression profile of M1- and M2-related genes in the spinal cord during the course of Theiler´s murine encephalomyelitis. A) Heat map displays fold changes, indicated by a color scale ranging from –4 (relative low expression) in green to 4 (relative high expression) in red. 59 out of 151 selected genes are differentially expressed in infected mice. B) Comparison of the relative proportion (percentage) of differentially
expressed M1- versus M2 marker genes employing the fisher´s exact test revealed a significant dominance (∗ =
p≤0.05) of M1-related genes at 14 and 42 days post infection (dpi). A statistical tendency (p = 0.078) of an increased M1-associated gene expression is observed at 98 dpi, whereas comparable proportions of M1- and M2-marker genes are detected at 196 dpi.
According to the function, differentially expressed genes were assigned to seven pathways, including chemotaxis (group I; 15 genes), phagocytosis, antigen processing and presentation (group II; 16 genes), cytokine and growth factor signaling (group III; 12 genes), Toll-like receptor signaling (group IV; 2 genes), apoptosis (Group V; 4 genes), extracellular matrix interaction and cell adhesion (group VI; 5 genes), and miscellaneous genes not related to a specific pathway (group VII; 5 genes; supplemental table S2). In group I, 53.3% of genes (8/15 genes) were up-regulated on 14 dpi, while at subsequent time points nearly all genes were significantly up-regulated. In group II and III 62.5% of genes (10/16 genes) and 50.0% of genes (6/12), respectively were up-regulated at 14 dpi, followed by an up-regulation of nearly all genes at 42, 98, and 196 dpi in both groups. Tlr1 (group IV) was significantly transcribed at 42, 98, and 196, while expression of Tlr2 was observed during the entire observation period. 75% of apoptosis-related genes (3/4 genes; group V) were significantly up-regulated in infected mice at 14 dpi and 100% at subsequent time points. While at 14 dpi 40.0% of genes (2/5 genes), all genes (100%) were up-regulated at 42, 98, and 196 dpi. Miscellaneous genes not assigned to a specific pathway (group VII) included Atf3, Arg1, Cepba, Chi3l3 and Hexb. No genes were differentially expressed at 14 dpi. Atf3, Arg1, and
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Cebpa were significantly increased at 42, 98 and 196 dpi, while the M2-marker Chi3l3 (aka Ym1) was only transcribed during the late chronic phase at 196 dpi (supplemental table S2). Immunofluorescence Immunofluorescence was used to confirm the results obtained by gene expression profiling. Employing the Spearman’s rank correlation coefficient, the amounts of all investigated macrophage/microglia proteins were significantly, positively correlated with the expression level of the respective genes (Table 1). The number of microglia/macrophages increased over time in the spinal cord of infected mice with highest numbers of CD107b+ and CD68+ microglia/macrophages in the late stages of the disease. CD16/32+ M1- and also arginase-1+ M2-type cells were significantly increased compared to non-infected animals at 42, 98 and 196 dpi (Figures 5 and 6).
Figure 5. Quantification of different macrophage/microglia subsets in the spinal cord of Theiler´s murine encephalomyelitis virus-infected mice by immunofluorescence. Significant increase of A) CD68+ cells, B) CD107b+ cells, C) arginase-1+ cells, and D) CD16/CD32+ cells in infected mice compared to mock-infected mice at 42, 98 and 196 days post infection. dpi = days post infection; mock = mock-infected control mice;
TMEV = Theiler´s murine encephalomyelitis virus-infected mice; ∗ = significant difference (p≤0.05, Mann-Whitney-U-test). Box and whisker plots display median and quartiles with maximum and minimum values.
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Figure 6. Detection of different macrophage/microglia subsets in the spinal cord of Theiler`s murine encephalomyelitis virus-infected mice by immunofluorescence. Accumulation of A) CD107b+ cells, B) CD68+ cells, C) arginase-1 (Arg-1)+ cells, and D) CD16/32+ cells in the spinal cord white matter at 196 days post infection. Inserts show higher magnifications of labelled cells. BIS = bisbenzimide (blue nuclear counterstain).
2.5 Discussion
The present study provides a comprehensive database of M1/M2-related gene-expression involved in the initiation and progression of TME. The onset of virus-induced demyelination is associated with a dominating M1-polarization, while mounting M2-polarization of macrophages/microglia together with sustained prominent M1-related gene expression are present during the chronic progressive phase. Differentially expressed M1-related genes at 14 dpi in the spinal cord of TMEV-infected mice predominately consist of factors, such as chemokines, involved in the CNS recruitment of macrophages, T cells and B cells (Table S2, group I). Simultaneously, migration of CD68+ antigen presenting cells and activation of genes related to innate and adaptive immunity within the CNS-draining cervical lymph node has been observed in TMEV-infected mice during the acute phase of the disease in our previous study (Navarrete-Talloni et al., 2010a). M1- responses are a hallmark of early innate immunity following viral infection mediated by the interaction between microglial toll-like receptors (Table S2, group IV) and cellular compounds (damage associated molecular pattern) and pathogen associated molecular pattern, respectively (Kigerl et al., 2009; Kigerl et al., 2007). However, besides their pivotal role for antiviral immunity, microglia have been demonstrated to induce also myelin-specific adaptive
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Th1-responses in TMEV-infected mice (Olson et al., 2001). Similarly, M1-polarized cells foster immunopathology in primary autoimmune CNS disorders (Mikita et al., 2011) and the drug Fasudil ameliorates the clinical severity of EAE by shifting macrophages/microglia from a M1- to a protective M2-phenotype (Liu et al., 2013). In addition, selective inhibition of M1-type microglia by minocycline treatment reduces neurodegeneration as demonstrated in mouse models for amyotrophic lateral sclerosis (Kobayashi et al., 2013). Similar to TME, experimental spinal cord injury in mice leads to microglial polarization into a pro-inflammatory and neurotoxic M1-phenotype, which might function as an early trigger of degeneration and immunological events at later disease stages (Kigerl et al., 2009). Excessive microglial responses can be observed also in human and canine spinal cord trauma, which leads to potentially destructive effects by the release of pro-inflammatory cytokines, proteolytic molecules and reactive oxygen species (Banati and Kreutzberg, 1993; Beineke et al., 2008; Ensinger et al., 2010; Markus et al., 2002; Qeska et al., 2013; Spitzbarth et al., 2011; Stein et al., 2008). Taken together, an imbalance towards M1-dominance represents a potential prerequisite for lesion initiation in TME as currently discussed for MS (Gandhi et al., 2010). Similar to findings in the present study, early innate immune responses with activated pro-inflammatory microglia can be detected in pre-demyelinating and early demyelinating MS lesions, which are supposed to induce myelin damage and immunopathology (Gandhi et al., 2010a; Marik et al., 2007). In the present study, the onset of demyelination and phagocytosis of myelin and apoptotic cells is accompanied by an up-regulation of genes involved in antigen processing, presentation and T cell stimulation (Table S2, group II). The functional relevance of phagocytic macrophages/microglia for the pathogenesis of CNS damage is discussed controversially. On the one hand, phagocytosis of myelin debris enhances CNS regeneration following traumatic injury (Yang and Schnaar, 2008). Moreover, ingestion of myelin induces a foamy appearance and anti-inflammatory function of cultured human macrophages and myelinophages within MS lesions acquire a M2-phenotype, which are supposed to contribute to resolution of inflammation and tissue repair (Boven et al., 2006). In addition, phagocytosis of apoptotic cells by cultured rodent microglia leads to diminished pro-inflammatory cytokine production with a reduced ability to activate T cells (Magnus et al., 2001). On the other hand, incorporation of myelin and cellular debris by microglia is able to enhance their antigen presenting and myelin-specific T cell stimulatory capacity in vitro (Beyer et al., 2000; Cash et al., 1993). Furthermore, isolated rat microglia exposed to myelin have been described to develop a neurotoxic phenotype with an increased inducible nitric oxide synthase, tumor necrosis factor-α and glutamate expression (Pinteaux-Jones et al., 2008) . Microarray analysis revealed the transcription of several genes participating in the interferon pathway predominately during the demyelinating phase (Table S2, group III). In TME, microglia/macrophages activated by virus or IFN-γ enhance immune mediated tissue damage by presenting viral antigens and endogenous myelin epitopes to CD4+ T cells, which induces delayed type hypersensitivity and autoimmunity, respectively (Borrow et al., 1992; Drescher et al., 1997; Katz-Levy et al., 2000; Pope et al., 1998). Moreover, beside its protective
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antiviral function, IFN-γ increases the migration of macrophages and microglial activation, which induces myelinotoxic substances and free radicals causing progressive myelin loss (bystander demyelination) in TME (Lipton, 1975; Miller et al., 2001; Tsunoda and Fujinami, 2002; Ulrich et al., 2006). Despite mounting M2-polarization and the expression of regeneration promoting factors, such as insulin like growth factor-1 (igf1) and transforming growth factor-β (Tgfb1) (Gudi et al., 2011; Lalive et al., 2005; Voss et al., 2012), CNS recovery remains abortive and only insufficient remyelination attempts by oligodendrocytes and Schwann cells were found in the spinal cord during the late chronic TME phase. Similar to the present observation, macrophages/microglia with both M1- and M2-properities can be found in active demyelinating MS brain lesions (Vogel et al., 2013). Recent studies have demonstrated that the switch of M1- into M2-type cells is required for efficient oligodendrocyte differentiation and myelin repair following toxin-induced demyelination in rodents and that M2-conditoned media drive oligodendrocyte maturation in vitro (Miron et al., 2013). In addition, M2-type macrophages/microglia protect from EAE through deactivation of encephalitogenic Th1 and Th17 cells (Qin et al., 2012). Consequently, continuous M1-polarization observed till the late chronic phase (196 dpi) in TMEV-infected mice has the potential to antagonize neuroprotective effects of M2-microglia/macrophages. Since TMEV has been demonstrated to preferentially infect activated myeloid cells with M1-charateristics, such as CD16/32 and IFN-γ expression, in vitro (Jelachich et al., 1999; Jelachich and Lipton, 1999), it is also tempting to speculate that prolonged M1-polarization contributes to viral persistence in susceptible mouse strains by providing permissive target cells for TMEV. In addition, genes have been identified by the present microarray analysis that might be involved in disturbed viral elimination by influencing the interferon pathway (Table S2, group III). For instance, OASL1, a recently defined type I interferon negative regulator and translation inhibitor of IRF7 is differentially up-regulated in TMEV-infected mice. OASL1 causes T cell suppression in persistent lymphocytic choriomeningitis virus infection of mice, and is regarded as a new target for preventing chronic infectious diseases (Lee et al., 2013; Leong et al., 2013). In agreement with this idea, subpopulations of CNS-infiltrating macrophages have been demonstrated to reduce protective antiviral immunity by inducing T cell exhaustion which leads to virus persistence in TMEV-infected mice (Jin et al., 2013). Besides this, M2-polarized cells have the ability to reduce antiviral immunity, as described for human cytomegalovirus infection (Avdic et al., 2013). In conclusion, the perpetuating interaction between virus and macrophages/microglia induces a vicious circle with continuous inflammation and impaired myelin repair in the spinal cord of TMEV-infected mice. The present findings support the hypothesis of a dual function of either polarized cells with promoting effects upon antiviral immunity and immunopathology, respectively, in TME. Hence, in contrast to the therapeutic effect of M2-dominence in primary autoimmune diseases, such as EAE, only a well-orchestrated and timely balanced polarization of macrophages/microglia might have the ability to prevent virus persistence and reduce myelin loss in this infectious MS model.
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2.6 Acknowledgements
The authors would like to thank Caroline Schütz, Kerstin Schöne, Danuta Waschke, Bettina Buck and Petra Grünig for their excellent technical support during the laboratory work and Dr. Karl Rohn for statistical analyses. This study was supported by the German Research Foundation (FOR 1103, BA 815/10-2, BE 4200/1-2 and UL 421/1-2). 2.7 References
Avdic S, Cao JZ, McSharry BP, Clancy LE, Brown R, Steain M, Gottlieb DJ, Abendroth A, Slobedman B (2013) Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses. Journal of virology 87:10273-10282.
Banati RB, Kreutzberg GW (1993) Flow cytometry: measurement of proteolytic and cytotoxic activity of microglia. Clinical neuropathology 12:285-288.
Beineke A, Markus S, Borlak J, Thum T, Baumgärtner W (2008) Increase of pro-inflammatory cytokine expression in non-demyelinating early cerebral lesions in nervous canine distemper. Viral immunology 21:401-410.
Bernard CC, de Rosbo NK (1991) Immunopathological recognition of autoantigens in multiple sclerosis. Acta neurologica 13:171-178.
Beyer M, Gimsa U, Eyupoglu IY, Hailer NP, Nitsch R (2000) Phagocytosis of neuronal or glial debris by microglial cells: upregulation of MHC class II expression and multinuclear giant cell formation in vitro. Glia 31:262-266.
Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185-193.
Borrow P, Tonks P, Welsh CJ, Nash AA (1992) The role of CD8+T cells in the acute and chronic phases of Theiler's murine encephalomyelitis virus-induced disease in mice. The Journal of general virology 73 ( Pt 7):1861-1865.
Boven LA, Van Meurs M, Van Zwam M, Wierenga-Wolf A, Hintzen RQ, Boot RG, Aerts JM, Amor S, Nieuwenhuis EE, Laman JD (2006) Myelin-laden macrophages are anti-inflammatory, consistent with foam cells in multiple sclerosis. Brain : a journal of neurology 129:517-526.
Cash E, Zhang Y, Rott O (1993) Microglia present myelin antigens to T cells after phagocytosis of oligodendrocytes. Cellular immunology 147:129-138.
Compston A, Coles A (2008) Multiple sclerosis. Lancet 372:1502-1517.
Dal Canto MC, Melvold RW, Kim BS, Miller SD (1995) Two models of multiple sclerosis: experimental allergic encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV) infection. A pathological and immunological comparison. Microscopy research and technique 32:215-229.
David S, Kroner A (2011) Repertoire of microglial and macrophage responses after spinal cord injury. Nature reviews Neuroscience 12:388-399.
Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s murine
encephalomyelitis
28
Drescher KM, Pease LR, Rodriguez M (1997) Antiviral immune responses modulate the nature of central nervous system (CNS) disease in a murine model of multiple sclerosis. Immunological reviews 159:177-193.
Durafourt BA, Moore CS, Zammit DA, Johnson TA, Zaguia F, Guiot MC, Bar-Or A, Antel JP (2012) Comparison of polarization properties of human adult microglia and blood-derived macrophages. Glia 60:717-727.
Ensinger E-M, Boekhoff TMA, Carlson R, Beineke A, Rohn K, Tipold A, Stein VM (2010) Regional topographical differences of canine microglial immunophenotype and function in the healthy spinal cord. Journal of neuroimmunology 227:144-152.
Gandhi R, Laroni A, Weiner HL (2010) Role of the innate immune system in the pathogenesis of multiple sclerosis. Journal of neuroimmunology 221:7-14.
Gerhauser I, Alldinger S, Baumgärtner W (2007) Ets-1 represents a pivotal transcription factor for viral clearance, inflammation, and demyelination in a mouse model of multiple sclerosis. Journal of neuroimmunology 188:86-94.
Gerhauser I, Hansmann F, Puff C, Kumnok J, Schaudien D, Wewetzer K, Baumgärtner W (2012) Theiler's murine encephalomyelitis virus induced phenotype switch of microglia in vitro. Journal of neuroimmunology 252:49-55.
Gudi V, Skuljec J, Yildiz O, Frichert K, Skripuletz T, Moharregh-Khiabani D, Voss E, Wissel K, Wolter S, Stangel M (2011) Spatial and temporal profiles of growth factor expression during CNS demyelination reveal the dynamics of repair priming. PloS one 6:e22623.
Haist V, Ulrich R, Kalkuhl A, Deschl U, Baumgärtner W (2012) Distinct Spatio-Temporal Extracellular Matrix Accumulation within Demyelinated Spinal Cord Lesions in Theiler's Murine Encephalomyelitis. Brain Pathology 22:188-204.
Herder V, Gerhauser I, Klein SK, Almeida P, Kummerfeld M, Ulrich R, Seehusen F, Rohn K, Schaudien D, Baumgärtner W, Huehn J, Beineke A (2012) Interleukin-10 expression during the acute phase is a putative prerequisite for delayed viral elimination in a murine model for multiple sclerosis. Journal of Neuroimmunology 249:27-39.
Hou W, Kang HS, Kim BS (2009) Th17 cells enhance viral persistence and inhibit T cell cytotoxicity in a model of chronic virus infection. The Journal of experimental medicine 206:313-328.
Jelachich ML, Bramlage C, Lipton HL (1999) Differentiation of M1 myeloid precursor cells into macrophages results in binding and infection by Theiler's murine encephalomyelitis virus and apoptosis. Journal of virology 73:3227-3235.
Jelachich ML, Lipton HL (1999) Restricted Theiler's murine encephalomyelitis virus infection in murine macrophages induces apoptosis. The Journal of general virology 80 ( Pt 7):1701-1705.
Jin YH, Hou W, Kang HS, Koh CS, Kim BS (2013) The role of interleukin-6 in the expression of PD-1 and PDL-1 on central nervous system cells following infection with Theiler's murine encephalomyelitis virus. Journal of virology 87:11538-11551.
Kakalacheva K, Munz C, Lunemann JD (2011) Viral triggers of multiple sclerosis. Biochimica et biophysica acta 1812:132-140.
Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s murine
encephalomyelitis
29
Katz-Levy Y, Neville KL, Padilla J, Rahbe S, Begolka WS, Girvin AM, Olson JK, Vanderlugt CL, Miller SD (2000) Temporal development of autoreactive Th1 responses and endogenous presentation of self myelin epitopes by central nervous system-resident APCs in Theiler's virus-infected mice. Journal of immunology 165:5304-5314.
Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. The Journal of neuroscience : the official journal of the Society for Neuroscience 29:13435-13444.
Kigerl KA, Lai W, Rivest S, Hart RP, Satoskar AR, Popovich PG (2007) Toll-like receptor (TLR)-2 and TLR-4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury. Journal of Neurochemistry 102:37-50.
Kobayashi K, Imagama S, Ohgomori T, Hirano K, Uchimura K, Sakamoto K, Hirakawa A, Takeuchi H, Suzumura A, Ishiguro N, Kadomatsu K (2013) Minocycline selectively inhibits M1 polarization of microglia. Cell death & disease 4:e525.
Kreutzer M, Seehusen F, Kreutzer R, Pringproa K, Kummerfeld M, Claus P, Deschl U, Kalkul A, Beineke A, Baumgärtner W, Ulrich R (2012) Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis. Brain pathology 22:454-471.
Kummerfeld M, Meens J, Haas L, Baumgärtner W, Beineke A (2009) Generation and characterization of a polyclonal antibody for the detection of Theiler's murine encephalomyelitis virus by light and electron microscopy. Journal of virological methods 160:185-188.
Kummerfeld M, Seehusen F, Klein S, Ulrich R, Kreutzer R, Gerhauser I, Herder V, Baumgärtner W, Beineke A (2012) Periventricular demyelination and axonal pathology is associated with subependymal virus spread in a murine model for multiple sclerosis. Intervirology 55:401-416.
Kumnok J, Ulrich R, Wewetzer K, Rohn K, Hansmann F, Baumgärtner W, Alldinger S (2008) Differential transcription of matrix-metalloproteinase genes in primary mouse astrocytes and microglia infected with Theiler's murine encephalomyelitis virus. Journal of neurovirology 14:205-217.
Lalive PH, Paglinawan R, Biollaz G, Kappos EA, Leone DP, Malipiero U, Relvas JB, Moransard M, Suter T, Fontana A (2005) TGF-beta-treated microglia induce oligodendrocyte precursor cell chemotaxis through the HGF-c-Met pathway. European journal of immunology 35:727-737.
Laskin DL (2009) Macrophages and inflammatory mediators in chemical toxicity: a battle of forces. Chemical research in toxicology 22:1376-1385.
Lee MS, Kim B, Oh GT, Kim YJ (2013) OASL1 inhibits translation of the type I interferon-regulating transcription factor IRF7. Nature immunology 14:346-355.
Lee MS, Park CH, Jeong YH, Kim YJ, Ha SJ (2013) Negative regulation of type I IFN expression by OASL1 permits chronic viral infection and CD8+ T-cell exhaustion. PLoS pathogens 9:e1003478.
Lipton HL (1975) Theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination. Infection and immunity 11:1147-1155.
Liu C, Li Y, Yu J, Feng L, Hou S, Liu Y, Guo M, Xie Y, Meng J, Zhang H, Xiao B, Ma C (2013) Targeting the shift from M1 to M2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil. PloS one 8:e54841.
Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s murine
encephalomyelitis
30
Liuzzi GM, Riccio P, Dal Canto MC (1995) Release of myelin basic protein-degrading proteolytic activity from microglia and macrophages after infection with Theiler's murine encephalomyelitis virus: comparison between susceptible and resistant mice. Journal of Neuroimmunology 62:91-102.
Magnus T, Chan A, Grauer O, Toyka KV, Gold R (2001) Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation. Journal of immunology 167:5004-5010.
Marik C, Felts PA, Bauer J, Lassmann H, Smith KJ (2007) Lesion genesis in a subset of patients with multiple sclerosis: a role for innate immunity? Brain : a journal of neurology 130:2800-2815.
Markus S, Failing K, Baumgärtner W (2002) Increased expression of pro-inflammatory cytokines and lack of up-regulation of anti-inflammatory cytokines in early distemper CNS lesions. Journal of neuroimmunology 125:30-41.
Martinez FO, Gordon S, Locati M, Mantovani A (2006) Transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression. Journal of immunology 177:7303-7311.
McMahon EJ, Bailey SL, Castenada CV, Waldner H, Miller SD (2005) Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nature medicine 11:335-339.
Mecha M, Carrillo-Salinas FJ, Mestre L, Feliu A, Guaza C (2013) Viral models of multiple sclerosis: neurodegeneration and demyelination in mice infected with Theiler's virus. Progress in neurobiology 101-102:46-64.
Mikita J, Dubourdieu-Cassagno N, Deloire MS, Vekris A, Biran M, Raffard G, Brochet B, Canron MH, Franconi JM, Boiziau C, Petry KG (2011) Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis. Amelioration of clinical status by M2 activated monocyte administration. Multiple sclerosis 17:2-15.
Miller SD, Olson JK, Croxford JL (2001) Multiple Pathways to Induction of Virus-Induced Autoimmune Demyelination: Lessons from Theiler's Virus Infection. Journal of Autoimmunity 16:219-227.
Miller SD, Vanderlugt CL, Begolka WS, Pao W, Yauch RL, Neville KL, Katz-Levy Y, Carrizosa A, Kim BS (1997) Persistent infection with Theiler's virus leads to CNS autoimmunity via epitope spreading. Nature medicine 3:1133-1136.
Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, van Wijngaarden P, Wagers AJ, Williams A, Franklin RJ, ffrench-Constant C (2013) M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nature neuroscience 16:1211-1218.
Monteyne P (1999) Infection virale du système nerveux central: du modèle expérimental à l'application humaine. Annales Françaises d'Anesthésie et de Réanimation 18:550-553.
Munz C, Lunemann JD, Getts MT, Miller SD (2009) Antiviral immune responses: triggers of or triggered by autoimmunity? Nature reviews Immunology 9:246-258.
Navarrete-Talloni MJ, Kalkuhl A, Deschl U, Ulrich R, Kummerfeld M, Rohn K, Baumgärtner W, Beineke A (2010) Transient peripheral immune response and central nervous system leaky compartmentalization in a viral model for multiple sclerosis. Brain pathology 20:890-901.
Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s murine
encephalomyelitis
31
Navarrete-Talloni MJ, Kalkuhl A, Deschl U, Ulrich R, Kummerfeld M, Rohn K, Baumgärtner W, Beineke A (2010) Transient Peripheral Immune Response and Central Nervous System Leaky Compartmentalization in a Viral Model for Multiple Sclerosis. Brain pathology 20:890-901.
Olson JK, Girvin AM, Miller SD (2001) Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler's virus. Journal of virology 75:9780-9789.
Ota K, Matsui M, Milford EL, Mackin GA, Weiner HL, Hafler DA (1990) T-cell recognition of an immuno-dominant myelin basic protein epitope in multiple sclerosis. Nature 346:183-187.
Pinteaux-Jones F, Sevastou IG, Fry VAH, Heales S, Baker D, Pocock JM (2008) Myelin-induced microglial neurotoxicity can be controlled by microglial metabotropic glutamate receptors. Journal of Neurochemistry 106:442-454.
Pope JG, Vanderlugt CL, Rahbe SM, Lipton HL, Miller SD (1998) Characterization of and functional antigen presentation by central nervous system mononuclear cells from mice infected with Theiler's murine encephalomyelitis virus. Journal of virology 72:7762-7771.
Prajeeth CK, Lohr K, Floess S, Zimmermann J, Ulrich R, Gudi V, Beineke A, Baumgärtner W, Muller M, Huehn J, Stangel M (2014) Effector molecules released by Th1 but not Th17 cells drive an M1 response in microglia. Brain, behavior, and immunity 37:248-259.
Qeska V, Barthel Y, Iseringhausen M, Tipold A, Stein VM, Khan MA, Baumgärtner W, Beineke A (2013) Dynamic changes of Foxp3(+) regulatory T cells in spleen and brain of canine distemper virus-infected dogs. Veterinary immunology and immunopathology 156:215-222.
Qin H, Yeh WI, De Sarno P, Holdbrooks AT, Liu Y, Muldowney MT, Reynolds SL, Yanagisawa LL, Fox TH, 3rd, Park K, Harrington LE, Raman C, Benveniste EN (2012) Signal transducer and activator of transcription-3/suppressor of cytokine signaling-3 (STAT3/SOCS3) axis in myeloid cells regulates neuroinflammation. Proceedings of the National Academy of Sciences of the United States of America 109:5004-5009.
Raddatz BB, Hansmann F, Spitzbarth I, Kalkuhl A, Deschl U, Baumgärtner W, Ulrich R (2014) Transcriptomic meta-analysis of multiple sclerosis and its experimental models. PloS one 9:e86643.
Rossi CP, Delcroix M, Huitinga I, McAllister A, van Rooijen N, Claassen E, Brahic M (1997) Role of macrophages during Theiler's virus infection. Journal of virology 71:3336-3340.
Skripuletz T, Miller E, Moharregh-Khiabani D, Blank A, Pul R, Gudi V, Trebst C, Stangel M (2010) Beneficial effects of minocycline on cuprizone induced cortical demyelination. Neurochemical research 35:1422-1433.
Spitzbarth I, Bock P, Haist V, Stein VM, Tipold A, Wewetzer K, Baumgärtner W, Beineke A (2011) Prominent microglial activation in the early proinflammatory immune response in naturally occurring canine spinal cord injury. Journal of neuropathology and experimental neurology 70:703-714.
Stein VM, Schreiner NMS, Moore PF, Vandevelde M, Zurbriggen A, Tipold A (2008) Immunophenotypical characterization of monocytes in canine distemper virus infection. Veterinary Microbiology 131:237-246.
Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. Proceedings of the National Academy of Sciences of the United States of America 100:9440-9445.
Tsunoda I (2008) Axonal degeneration as a self-destructive defense mechanism against neurotropic virus infection. Future virology 3:579-593.
Dynamic changes of microglia/macrophage M1 and M2 polarization in Theiler’s murine
encephalomyelitis
32
Tsunoda I, Fujinami RS (1996) Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus. Journal of neuropathology and experimental neurology 55:673-686.
Tsunoda I, Fujinami RS (2002) Inside-Out versus Outside-In models for virus induced demyelination: axonal damage triggering demyelination. Springer seminars in immunopathology 24:105-125.
Ulrich R, Baumgärtner W, Gerhauser I, Seeliger F, Haist V, Deschl U, Alldinger S (2006) MMP-12, MMP-3, and TIMP-1 are markedly upregulated in chronic demyelinating theiler murine encephalomyelitis. Journal of neuropathology and experimental neurology 65:783-793.
Ulrich R, Kalkuhl A, Deschl U, Baumgärtner W (2010) Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis. Journal of cellular and molecular medicine 14:434-448.
Ulrich R, Seeliger F, Kreutzer M, Germann PG, Baumgärtner W (2008) Limited remyelination in Theiler's murine encephalomyelitis due to insufficient oligodendroglial differentiation of nerve/glial antigen 2 (NG2)-positive putative oligodendroglial progenitor cells. Neuropathology and Applied Neurobiology 34:603-620.
Vogel DY, Vereyken EJ, Glim JE, Heijnen PD, Moeton M, van der Valk P, Amor S, Teunissen CE, van Horssen J, Dijkstra CD (2013) Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status. Journal of neuroinflammation 10:35.
Voss EV, Skuljec J, Gudi V, Skripuletz T, Pul R, Trebst C, Stangel M (2012) Characterisation of microglia during de- and remyelination: can they create a repair promoting environment? Neurobiology of disease 45:519-528.
Yang LJS, Schnaar RL (2008) Axon regeneration inhibitors. Neurological research 30:1047-1052.
Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant
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3. Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice
Chittappen K. Prajeeth†,1, Andreas Beineke§,#,1, Cut Dahlia Iskandar§,#, Viktoria Gudi†, Vanessa Herder§, Ingo Gerhauser§, Verena Haist§, René Teich‡, Jochen Huehn‡, Wolfgang Baumgärtner§,#, Martin Stangel†,#
† Clinical Neuroimmunology and Neurochemistry, Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany § Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany ‡ Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, D-
38124 Braunschweig, Germany # Center of Systems Neuroscience
1 contributed equally to this work; Key words: regulatory T cells; Theiler virus; interleukin-10 Contribution of the PhD student: C.D. Iskandar performed immunohistochemistry and contributed to the analyses of immunofluorescence microscopy and statistical analyses. Correspondence: Prof. Dr. Martin Stangel Clinical Neuroimmunology and Neurochemistry Department of Neurology Hannover Medical School Carl-Neuberg-Str. 1 30625 Hannover Germany Ph. +49-511-532 6676 Fax +49-511-532 3115 E-mail: [email protected]
Journal of Neuroinflammation http://www.jneuroinflammation.com
DOI: 10.1186/s12974-014-0180-9
Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant
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Abstract Background Theiler’s murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3+ CD4+ regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse stains. Methods In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3+ Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler’s murine encephalomyelitis (TME). Results As expected, DEREG mice which are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of interleukin-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells. Conclusions We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.
General discussion
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4. GENERAL DISCUSSION 4.1 Disease phase-specific changes of macrophages/microglia polarization in
Theiler’s murine encephalomyelitis The aim of the present study was to investigate changes of microglia/macrophage polarization at different phases of TME, a model for chronic viral CNS infections and myelin loss disorders, respectively (Oleszak et al., 2004). Microglia and CNS-infiltrating macrophages have been demonstrated to contribute to the initiation and progression of demyelination in the spinal cord white matter by antigen presentation, inducing cytotoxicity and regulating T and B cell responses (Aloisi, 2001; Mecha et al., 2013). Results of the first part of the project demonstrate the occurrence of phase dependent differences of macrophage/microglia subsets in TME. During the predemyelinating TME phase an early activation of microglia, associated with a dominant M1-polarization was observed. Similarly, an activation of glial cells, particularly microglia, has been described in early MS lesions (Gandhi et al., 2010; Marik et al., 2007). Although the role of microglia in MS is discussed controversially, the importance of microglia for the initiation of myelin damage has been demonstrated in a variety of experimental and spontaneous CNS disorders. For instance, an early activation of resident glial cells together with a predominant pro-inflammatory cytokine environment, indicative potentially neurotoxic M1-microglial polarization can be observed in canine distemper (Beineke et al., 2008). This hypothesis is supported by the observation of an increased expression of adhesion molecules, phagocytic activity and release of reactive oxygen species of microglia in canine distemper virus (CDV) infected dogs (Stein et al., 2004; Stein et al., 2008). M1 microglial responses and release of myelinotoxic factors also contribute to lesion development in EAE (Mikita et al., 2011).
Similar to primarily inflammatory disorders, induction of neurotoxic M1-type microglial cells is supposed to represent a prerequisite for subsequent immune mediated tissue damage following spinal cord injury in dogs (Spitzbarth et al., 2011). A dominating M1 polarization can be detected during the early lesion development in rodent models for spinal cord injury (Goldmann and Prinz, 2013; Kigerl et al., 2009). Besides these effects upon myelin sheaths and oligodendrocytes, an early activation of microglia has been shown also in the cerebral grey matter during the early TME phase, leading to acute polioencephalitis (Boche et al., 2013; Mecha et al., 2013; Rossi et al., 1997). This early process is influenced by adenosine triphosphate (ATP) release and purinergic receptor engagement in focal brain lesions (Giunti et al., 2014), associated with an increased phagocytic activity and cytokine transcription of microglia (Gerhauser et al., 2012). Prominent M1 polarization is often related to neuronal damage in infectious CNS diseases (Goldmann and Prinz, 2013), initiated by TLRs interaction (Giunti et al., 2014).
General discussion
36
Infection of intralesional microglia/macrophages contributes to virus persistence and lesion progression in TMEV-infected mice (Kim et al., 2005a; Rossi et al., 1997). As described previously, an increasing amount and activity of microglia/macrophages have been observed during the disease course in the present survey (Ulrich et al., 2010). Similarly, continuous CDV-induced microglial activation is supposed to cause progressive demyelination (bystander demyelination) in affected dogs (Botteron et al., 1992; Griot et al., 1990). Strikingly, an imbalance between M1 and M2 cells have been detected in TMEV infected SJL mice with a disproportional switch towards M1 polarization from 42 dpi until 196 dpi. These results are in agreement with previous reports demonstrating an increased activity of pro-inflammatory genes during the chronic phase of TME (Ulrich et al., 2010). The M1 dominance is supposed to trigger pro-inflammatory and neurotoxic processes in the spinal cord of TMEV-infected animals (David and Kroner, 2011; Durafourt et al., 2012). Highest numbers of M1 and M2 cells and gene expressions have been detected at 98 dpi in the present study which correlates with neuroinflammation and demyelination in these animals. Topographical differences of glial cell functionality might be responsible for different sensitivities to injury of the white matter in brain and spinal cord of TMEV-infected SJL mice (Kummerfeld et al., 2012). A reduced myelin degrading proteolytic activity of microglia and macrophages might explain the lack of demyelination in C57BL/6 mice (Liuzzi et al., 1995b). In analogy to the concept of region-specific lesion development in TME and human MS, recent studies have demonstrated topographical differences of de- and remyelination within the brain of cuprizone fed mice, which might be partly attributed to unequal densities or functions of microglia in different CNS compartments (Gudi et al., 2009; Skripuletz et al., 2008; Skripuletz et al., 2010). Usually a switch from M1 to M2 polarization leads to termination of inflammatory responses in the CNS (David and Kroner, 2011), which was not observed in the present survey. Microglial cells are able to gain a M2 phenotype characterized by arginase-1 and IL-10 expression (Goldmann and Prinz, 2013; Rawji and Yong, 2013). However, reduced expression of M1 cells is essential for maintaining M2 activation. Thus, prolonged M1 responses might contribute to inadequate or delayed M2 responses and TMEV-induced myelin loss, respectively (David and Kroner, 2011; Laskin, 2009). 4.2 Effects of macrophages/microglia polarization upon regeneration in the central
nervous system Despite ongoing spinal cord inflammation and demyelination, transmission electron microscopy revealed the occurrence of remyelination by Schwann cells and oligodendrocytes during the late chronic phase of TME (196 dpi) in the present study. Macrophages/microglia have the ability to secrete factors that stimulate axonal regrowth and oligodendrocyte differentiation (Diemel et al., 1998). Moreover, since myelin debris inhibits remyelination, in addition to the trophic function mediated by M2-type cells, removal of necrotic tissue by phagocytic macrophages/microglia are required for adequate CNS regeneration (Miron and Franklin, 2014). Generally, M2a promote tissue repair and M2b cells as well as M2c exhibit
General discussion
37
debris scavenger function. In the present study a predominant activation of M2c-related genes has been observed (data not shown), probably activated by TGF-β and IL-10. These cells have the ability to down-regulate M1 responses, induce high levels of arginase and promote wound healing, tissue remodelling and angiogenesis (David and Kroner, 2011; Laskin, 2009). Phagocytosis, collagen formation and Th2 cell recruitment is promoted by M2a cells (David and Kroner, 2011; Laskin, 2009). The role of microglia and macrophages in myelin repair and myelinogenesis has been demonstrated in in vitro experiments (Diemel et al., 1998). Here, microglia-derived factors increase the expression of myelin-specific genes, such as MBP, proteolipid protein and myelin associated glycoprotein in dissociated brain cultures (Hamilton and Rome, 1994; Loughlin et al., 1997). Recent in vivo studies have shown that dominant M2-cell responses are required for efficient oligodendrocyte differentiation and myelin repair following toxin-induced (lysolecithin; ethidium bromide) demyelination in rodents. Moreover, M2-conditoned media drives oligodendrocyte maturation with an enhanced expression of MBP and MOG in vitro (Miron et al., 2013). Also, activin-A, a member of the TGF-β superfamily contributes to the regenerative function of M2 macrophages (Miron et al., 2013; Miron and Franklin, 2014). Other trophic factors released by macrophages and/or microglia include platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), TGF-β, insulin-like growth factor 1 (IGF-1), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophic factor-3 (NT-3) (Diemel et al., 1998). In the present study, despite mounting M2-polarization and the expression of regeneration promoting factors, such as IGF-1, remyelination remains abortive in the late chronic TME phase which supports the pivotal role of a switch from M1- into M2-cells for efficient oligodendrocyte differentiation and myelin repair also in this infectious model for demyelinating diseases. An inappropriate switch from M1 to M2 macrophages/microglia is also a cause for delayed oligodendrocyte differentiation in ageing (Miron et al., 2013). In addition to the effect upon remyelination, activated rat M2-microglia have been demonstrated to increase neuronal differentiation in vitro, mediated by protease serine 2 (Nikolakopoulou et al., 2013). In agreement with this, minocycline induced modulation of microglia reduce neuronal degeneration and improves neurogenesis in rodent model of schizophrenia by reducing the expression of TNF-α and IL-1β in the hippocampus (Mattei et al., 2014). Similar to MS, limited remyelination due to dysfunctional oligodendrocyte precursor cells has been described in the murine spinal cord following TMEV infection (Ulrich et al., 2008). Additionally, maturation and differentiation of an oligodendrocyte precursor cell line has been shown to be impaired by TMEV in vitro (Pringproa et al., 2010). Thus, prolonged myelin degradation and impaired myelin repair in the spinal cord white matter observed in the present study might be sequel of dominant M1-responses and insufficient M2 polarization of macrophages/microglia during the late chronic phase of TME.
General discussion
38
4.3 Interaction between regulatory T cells and other immune cells of the central nervous system
Treg play a key role in the maintenance of immunological tolerance (immune privilege status of the CNS) and prevent immunopathology in various systemic and CNS diseases (Feuerer et al., 2010; Feuerer et al., 2009; MacDonald et al., 2002; Sakaguchi, 2003; Sakaguchi et al., 2006; Vignali et al., 2008). For instance, depletion of Treg leads to an activation of auto-aggressive T cells, while the adoptive transfer or in vivo expansion of Treg reduce immune mediated demyelination in EAE (Jee et al., 2007; Korn et al., 2007). Moreover, functional impairment or an insufficient amount of Treg is supposed to contribute to demyelination in acute MS lesions (Fritzsching et al., 2011). Neuroprotective functions of Treg can be observed also in degenerative CNS disorders, such as stroke (Liesz et al., 2009). However, in viral diseases these cells can exhibit both beneficial effects by reducing immune mediated tissue damage and detrimental effects due to their immunosuppressive properties, causing disease exacerbation or viral persistence, respectively (Gobel et al., 2012; Lund et al., 2008). Recently, rapid expansion of Treg associated with an increased expression of the immunosuppressive cytokine IL-10 has been demonstrated in the brain of susceptible SJL mice but not in resistant C57BL/6 mice following TMEV infection (Herder et al., 2012a; Richards et al., 2011). These results demonstrate the important effect of Treg upon virus-specific immunity. Besides naturally occurring Foxp3+ Treg, inducible Treg including Tregulatory-1 (Tr1) cells, Th3 cells and CD8+ Treg can be observed in CNS disorders (Lowther and Hafler, 2012). For instance, Tr1 cells secrete increased amounts of IL-5, IL-10 and TGF-ß and develop from naïve CD4+ T cells due to chronic stimulation in infectious or neoplastic diseases (Fletcher et al., 2010; Nandakumar et al., 2009). Thus, in addition to the migration of natural Treg from peripheral lymphoid organs, the inflammatory environment might influence the balance of Treg versus effector T cell differentiation which might lead to local de novo induction and expansion of Foxp3+ Treg within the brain of TMEV-infected animals. Referring to this, virus-specific CD4+ T cells can acquire a Treg phenotype including Foxp3 expression in the CNS, as described for experimental mouse hepatitis virus infection (Zhao et al., 2011). Moreover, neurons and activated astrocytes are able to induce Treg to decrease excessive inflammation and demyelination in EAE (Liu et al., 2006; Trajkovic et al., 2004). Strikingly, Foxp3, which was shown to represent a key transcription factor of Treg can also be observed in activated microglia with immunomodulatory properties (Chung et al., 2010). Suppression of effector T cells, including Th17 cells, by Treg, is mediated by their secretion of the inhibitory cytokines IL-10, TGF-β and IL-35 or expression of the ectoenzymes CD39 and CD73. In addition, Treg can induce apoptosis of effector T cells by granzyme release or IL-2 deprivation, respectively. Indirect mechanisms of T cell suppression include disturbed maturation and antigen presenting function of dendritic cells via Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4) ligation by Treg (Vignali et al., 2008). In addition, Foxp3+ Treg have the ability to induce a M2 phenotype of microglia and macrophages which exhibit immunomodulatory properties and promote regeneration in the injured CNS (Chung et al.,
General discussion
39
2010; Huang et al., 2010) (Beers et al., 2011; Tiemessen et al., 2007). Recruitment of Treg to the brain is mediated by M2a and M2b cells (David and Kroner, 2011; Laskin, 2009). They are able to attenuate microglial cytotoxicity through cell-to-cell contact, which leads to the protection of motoneurons (Zhao et al., 2012). Treg-associated M2-polarization leads to an up-regulation of BDNF and glial cell-derived neurotrophic factor (GDNF) expression and down-regulation of pro-inflammatory cytokines and oxidative stress (Liu et al., 2009). CNS-infiltrating Treg have the capacity to reduce glial responses, including astrogliosis, as observed in animal models of stroke and experimental autoimmune encephalomyelitis (EAE) (Beyersdorf et al., 2005; Liesz et al., 2009), as well as in human immunodeficiency virus-1-associated neurodegeneration (Liu et al., 2009). On the other hand, activated astrocytes are able to induce Treg to decrease excessive inflammation and demyelination in EAE (Liu et al., 2006; Trajkovic et al., 2004). Moreover, loss of astrocytes in GFAP-Cre gp130fl/fl mice results in a reduction of Foxp3+ Treg and an increase of IL-17-, IFN-γ- and TNF-producing effector T cells in EAE, demonstrating a regulatory function of astrocytes (Haroon et al., 2011). Furthermore, as demonstrated in GFAP-Cre FasLfl/fl mice, astrocytes induce Fas Ligand-mediated apoptotic elimination of encephalitogenic T cells but not of protective Treg in order to recover from EAE (Wang et al., 2013). Dendritic cells increase within the CNS as a consequence of inflammation associated with a variety of autoimmune and infectious diseases (D'Agostino et al., 2012; Zozulya et al., 2009). Stimulatory dendritic cells exacerbate the severity of EAE, accompanied with an early infiltration of effector T cells and reduced proportions of Foxp3+ Treg in the brain, while semi mature dendritic cells induce immune tolerance and attenuate the disease course (Zozulya et al., 2009). In contrast to this beneficial effect of tolerogenic dendritic cells and Treg in this autoimmune model, Japanese encephalitis virus-infected dendritic cells expand Treg by increasing PD-Ligand1 expression, representing a potential mechanism of the pathogen to evade host immune responses (Gupta et al., 2014). Results of the present project are in good agreement with previous observations demonstrating that functional inactivation of Treg by anti-CD25-antibodies and the adoptive transfer of Treg failed to influence the disease course in resistant mice strains (C57BL/6), demonstrating the complexity of Treg function in infectious CNS disorders and the pivotal role of CD8-mediated cytotoxicity for TMEV elimination (Martinez et al., 2014; Richards et al., 2011). Similarly, indoleamine 2,3 dioxygenase (IDO) expression of brain tumor-infiltrating dendritic cells potentially increases the CNS recruitment of Treg, which reduces antitumoral immune responses and decreases the survival of patients with glioblastoma multiforme (Wainwright et al., 2012; Wainwright et al., 2014).
Conclusions
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5. CONCLUSIONS
In conclusion, the perpetuating interaction between virus and microglia/macrophages induces
a vicious circle with continuous inflammation and impaired myelin repair in the spinal cord of
TMEV-infected mice. The present findings support the hypothesis of a dual function of cells
of both polarities with promoting effects upon antiviral immunity and immunopathology,
respectively, in TME. Modulating the microglia polarization of the spinal cord might
represent a prerequisite to stimulate endogenous regeneration and future transplantation
approaches (Kobayashi et al., 2013). However, since M1-type cells are pivotal for virus
elimination, in contrast to primarily autoimmune CNS disorders, reconstitution of
immunomodulatory microglia/macrophages might be necessary, rather than simple
suppression of M1-responses to establish CNS recovery in this infectious MS model. Hence,
in contrast to the therapeutic effect of M2-dominence in primary autoimmune diseases, such
as EAE, only a well-orchestrated and timely balanced polarization of macrophages/microglia
might have the ability to prevent virus persistence and reduce myelin loss in this infectious
MS model.
Summary
41
6. SUMMARY Cut Dahlia Iskandar Polarization of immune cells in Theiler’s murine encephalomyelitis Multiple sclerosis (MS), one of the most frequent central nervous system (CNS) diseases in young adults, is a chronic demyelinating disease of unknown etiology and possibly multifactorial causes. Microglia and macrophages play a central role for demyelination in Theiler’s murine encephalomyelitis (TME) virus-infection, a commonly used viral mouse model for the chronic-progressive form of MS. Microglia and CNS-infiltrating macrophages play a central role in the pathogenesis of TME virus-induced demyelination, e.g. as target cells for viral persistence. Similar to other demyelination models, such as experimental autoimmune encephalomyelitis (EAE), they also induce bystander demyelination, delayed-type hypersensitivity and myelin-specific autoimmunity. The current concept of microglia/macrophages plasticity describes different cell populations with distinct and even opposing functions. For instance, M1-type microglia/macrophages exhibit pro-inflammatory properties, while M2-type cells exhibit neuroprotective properties. However, so far, only few reports mention the polarizing effects of TME virus upon microglia/macrophages. Therefore, the aim of the present project was to determine dynamic changes of microglia/macrophage polarization in the spinal cord of susceptible SJL mice during the initiation and progression of TME. Moreover, the relevance of regulatory T cells (Treg) for polarization of immune cells, including microglia/macrophages, was investigated by genetic ablation of Foxp3+ Treg in resistant C57BL/6 mice following TME virus infection. In the first part of the study, the spinal cord of TME virus-infected SJL mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and chronic demyelinating leukomyelitis was confirmed by immunohistochemistry and histology, respectively. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase, indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TMEV-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1-polarization, while mounting M2-polarization of macrophages/microglia together with sustained prominent M1-related gene expression were present during the chronic progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32+ M1- and arginase-1+ M2-type cells associated with progressive demyelination. The study provides a comprehensive database of M1/M2-related gene expression involved in the initiation and progression of demyelination, which supports the hypothesis that the perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.
Summary
42
The second part of the study aimed to gain further insights into the relevance of Treg for disease resistance and antiviral immunity in TME, the kinetics of CNS immune cells and the underlying chemokine and cytokine expression following genetic ablation of Treg in BAC-transgenic Foxp3 reporter mice (DEREG mice) with a C57BL6 background. As determined by RT-qPCR, DEREG mice were resistant to TME virus infection and cleared the virus, regardless of the presence or absence of Treg. Nevertheless, priming of strong effector T cell responses was observed in the periphery following Treg ablation, which subsequently resulted in a transient increase of IFNγ-producing T cells in the brain. Histology, Immunohistochemistry and flow cytometric analysis revealed that this transient increase of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice was not associated with an augmented antiviral response or increased inflammation-mediated tissue damage, respectively. Expression of interleukin-10 in the infected brain was unaltered despite of Treg depletion, which might play a role for dampening the inflammatory damage caused by increased number of effector T cells. Thus, unlike susceptible SJL mice, Treg have only negligible effects on virus-induced pathologies in the CNS of the resistant C57BL/6 mice. In conclusion, the present findings of the first part support the hypothesis of a dual function of microglia/macrophges with promoting effects upon antiviral immunity and immunopathology, respectively. Modulating the microglia polarization of the spinal cord might represent a prerequisite to stimulate endogenous regeneration and future transplantation approaches. However, since M1-type cells are pivotal for virus elimination, in contrast to primarily autoimmune CNS disorders, reconstitution of immunomodulatory microglia/macrophages might be necessary, rather than simple suppression of M1-responses to establish CNS recovery in this infectious MS model. Hence, in contrast to the therapeutic effect of M2-dominence in primary autoimmune diseases, such as EAE, only a well-orchestrated and timely balanced polarization of macrophages/microglia might have the ability to prevent virus persistence and reduce myelin loss in this infectious MS model. Results of the second part confirm that resistance in C57BL/6 mice to TME virus infection is largely due to the induction of effective CD4+ and CD8+ T cell responses, which is not significantly influenced by Treg depletion. Sustained expression of interleukin-10, probably by neuroprotective M2-type microglia/macrophages during early infection, might compensate for the lack of Treg and limit the extent of damage caused by an unwanted immune response in the brain.
Zusammenfasung
43
7. ZUSAMMENFASSUNG
Cut Dahlia Iskandar Polarisation von Immunzellen bei der Theilerschen murinen Enzephalomyelitis Bei der Mulitplen Sklerose des Menschen handelt es sich um eine der häufigsten Erkrankungen des zentralen Nervensystems (ZNS) bei jungen Erwachsenen. Die Erkrankung führt zu einer chronisch-entzündlichen Entmarkung, wobei die primäre Ursache bislang ungeklärt ist. Die demyelinisierende murine Theilervirus-Enzephalomyelitis (TME) stellt ein sehr gutes und viral-induziertes Modell für MS dar. Mikroglia und Makrophagen spielen bei der Entmarkung der TME eine wichtige Rolle, insbesondere als Zielzellen während der chronischen Phase, wodurch es zur Viruspersistenz kommt. Außerdem rufen diese Zellen bei MS und TME, wie auch bei anderen Entmarkungskrankheiten (z.B. experimentelle autoimmune Enzephalomyelitis [EAE]) Hypersensitivitätsreaktionen vom verzögerten Typ und eine myelinspezifische Autoimmunität hervor und setzen myelintoxische Substanzen frei (bystander demyelination). Man geht derzeit davon aus, dass die Mikroglia/Makrophagen-Population zwei unterschiedliche, zum Teil gegensätzliche Funktionen aufweisen. Dabei wird zwischen den Mikroglia/Makrophagen vom M1- und M2-Typ unterschieden. M1-Mikroglia/Makrophagen weisen pro-inflammatorische Eigenschaften auf, während die Mikroglia/Makrophagen vom M2-Typ neuroprotektive Eigenschaften zeigen. Bisher gibt es nur wenige Daten zur Mikroglia/Makrophagen-Polarisierung im Verlauf der TME. Aus diesem Grund war es Ziel dieser Studie, die Veränderungen der Mikroglia/Makrophagen-Polarisierung im Rückenmark von virusinfizierten, empfänglichen SJL-Mäusen im Initialstadium der TME und zu späteren Zeitpunkten der progressiven Erkrankung zu untersuchen. Weiterhin wurde die Bedeutung des Einflusses von regulatorischen T Zellen (Treg) auf die Immunzellen, inklusive Mikroglia/Makrophagen, bei der TME untersucht. Im ersten Teil dieser Studie wurde das Rückenmark von TME-Virus-infizierten SJL-Mäusen mittels Genexpressionsanalyse und Immunfluoreszenz untersucht. Darüber hinaus wurden die Viruspersistenz und die chronisch-demyelinisierende Leukomyelitis mittels Immunhistochemie und Histologie dargestellt. Die elektronenmikroskopische Untersuchung ergab einen kontinuierlichen Myelinverlust, der mit abortiven Reparaturmechanismen bzw. einer unvollständigen Remyelinisierung während der chronischen Infektionsphase einherging. Für die Genexpressionsanalyse im Rückenmark von TME-Virus-infizierten SJL-Mäusen wurden 151 Gene ausgewählt, von denen 59 differentiell exprimiert waren. Zu Beginn der virusinduzierten Demyelinisierung wurde eine dominante M1-Polarisierung festgestellt. Die Spätphase der Demyelinisierung war durch eine Zunahme der M2-polarisierten Mikroglia/Makrophagen charakterisiert, die allerdings weiterhin von einer dominierenden M1-Polarisierung begleitet war. Die Ergebnisse der Genexpressionsanalyse wurden durch die Resultate der Immunfluoreszenz verifiziert. Hierbei zeigte sich ein vermehrter Nachweis von CD16/32+ M1- und Arginase-1+ M2-polarisierten Zellen in der chronisch-progressiven Phase. Diese Studie stellt umfassende Daten zur Expression von M1- und M2-assoziierten Genen
Zusammenfasung
44
während der Initiation und Progression der TME-Virus-induzierten Demyelinisierung zur Verfügung. Darüber hinaus unterstützen die Ergebnisse die Hypothese, dass die kontinuierliche Interaktion zwischen dem Erreger und Mikroglia/Makrophagen für die persistente Entzündung und Remyelinisierungsstörung bei der TME mitverantwortlich ist. Im zweiten Teil der Studie sollte die Bedeutung der Treg für Mechanismen der TME-Resistenz und der antiviralen Immunantwort bei C57BL/6-Mäusen näher untersucht werden. Hierfür wurde die Immunzellinfiltration sowie das zugrunde liegende Chemokin- und Zytokinexpressionsprofil in TME-Virus-infizierten BAC-transgenen Foxp3-Reporter-Mäusen (DEREG-Mäuse) mit einem C57BL/6-Hintergrund untersucht. Unabhängig vom Vorhandensein von Treg wurde mittels RT-qPCR gezeigt, dass DEREG-Mäuse resistent gegenüber der TME-Virus-Infektion sind und das Virus eliminieren. In peripheren lymphatischen Zellen konnte eine deutliche Effektor-T Zellantwort nach Ablation der Treg in infizierten DEREG-Mäusen festgestellt werden. Dies resultierte in einer transienten Zunahme IFNγ-produzierender T-Zellen im Gehirn. Mittels Histologie, Immunhistochemie und
Durchflusszytometrie wurde festgestellt, dass dieser transiente Anstieg IFNγ-produzierender T-Zellen bei Treg-depletierten DEREG-Mäusen nicht mit einer gesteigerten antiviralen Immunantwort oder einem deutlicherem Gewebeschaden im Gehirn vergesellschaftet war. Die Expression von Interleukin-10 war trotz der Treg-Ablation im Gehirn von virusinfizierten Mäusen unverändert, was für einen potentiell neuroprotektiven Effekt des Zytokins bei der TME spricht. Folglich haben Treg in TME-resistenten C57BL/6-Mäusen einen nur sehr geringen Effekt auf die virusinduzierten pathologischen Veränderungen im ZNS, im Gegensatz zu TME-empfänglichen SJL-Mäusen. Zusammenfassend weisen die Ergebnisse der ersten Studie darauf hin, dass Mikroglia und Makrophagen eine duale Funktion besitzen und sowohl die antivirale Immunantwort als auch die Immunpathologie bei der TME beeinflussen. Die Modulation der Makrophagen/Mikroglia-Polarisation im Rückenmark könnte eine Methode zur Stimulation der endogenen Regeneration und eine adjuvante Therapiestrategie bei zukünftigen Transplantations-Studien darstellen. Im Gegensatz zu primär autoimmunen Erkrankungen des ZNS (z.B. EAE) spielen bei der TME M1-polarisierte Mikroglia/Makrophagen anscheinend eine große Rolle für die Viruselimination. Aus diesem Grund muss daher die Abfolge der M1- und M2-induzierten Effekte ausbalanciert und zeitlich abgestimmt sein, um die Viruspersistenz zu vermeiden und um den Myelinverlust zu limitieren. Die Ergebnisse des zweiten Teils dieser Studie konnten zeigen, dass die Resistenz der C57BL/6-Mäuse gegenüber der TME weitestgehend auf einer Induktion von CD4+ und CD8+ T Zellen beruht, die nicht signifikant von der Treg-Ablation beeinflusst wird. Eine anhaltende Interleukin-10-Expression, möglicherweise durch Mikroglia/Makrophagen vom M2-Typ während der Frühphase der Infektion, kompensiert hierbei möglicherweise den Treg-Mangel und limitiert den Gewebsschaden.
References
45
8. REFERENCES
Aguzzi, A., Barres, B.A., Bennett, M.L., 2013. Microglia: scapegoat, saboteur, or something else? Science 339, 156-161.
Aloisi, F., 2001. Immune function of microglia. Glia 36, 165-179.
Amor, S., Puentes, F., Baker, D., Van Der Valk, P., 2010. Inflammation in neurodegenerative diseases. Immunology 129, 154-169.
Andersson, A., Karlsson, J., 2004. Genetics of experimental autoimmune encephalomyelitis in the mouse. Archivum immunologiae et therapiae experimentalis 52, 316-325.
Andjelkovic, A.V., Nikolic, B., Pachter, J.S., Zecevic, N., 1998. Macrophages/microglial cells in human central nervous system during development: an immunohistochemical study. Brain research 814, 13-25.
Barnett, M.H., Prineas, J.W., 2004. Relapsing and remitting multiple sclerosis: pathology of the newly forming lesion. Annals of neurology 55, 458-468.
Baumann, N., Pham-Dinh, D., 2001. Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiological reviews 81, 871-927.
Baxter, A.G., 2007. The origin and application of experimental autoimmune encephalomyelitis. Nature reviews. Immunology 7, 904-912.
Beers, D.R., Henkel, J.S., Zhao, W., Wang, J., Huang, A., Wen, S., Liao, B., Appel, S.H., 2011. Endogenous regulatory T lymphocytes ameliorate amyotrophic lateral sclerosis in mice and correlate with disease progression in patients with amyotrophic lateral sclerosis. Brain : a journal of neurology 134, 1293-1314.
Beineke, A., Markus, S., Borlak, J., Thum, T., Baumgärtner, W., 2008. Increase of pro-inflammatory cytokine expression in non-demyelinating early cerebral lesions in nervous canine distemper. Viral immunology 21, 401-410.
Beineke, A., Puff, C., Seehusen, F., Baumgärtner, W., 2009. Pathogenesis and immunopathology of systemic and nervous canine distemper. Veterinary immunology and immunopathology 127, 1-18.
Beyersdorf, N., Gaupp, S., Balbach, K., Schmidt, J., Toyka, K.V., Lin, C.H., Hanke, T., Hunig, T., Kerkau, T., Gold, R., 2005. Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis. The Journal of experimental medicine 202, 445-455.
Black, J.A., Waxman, S.G., 2012. Sodium channels and microglial function. Experimental neurology 234, 302-315.
Boche, D., Perry, V.H., Nicoll, J.A.R., 2013. Review: Activation patterns of microglia and their identification in the human brain. Neuropathology and applied neurobiology 39, 3-18.
Botteron, C., Zurbriggen, A., Griot, C., Vandevelde, M., 1992. Canine distemper virus-immune complexes induce bystander degeneration of oligodendrocytes. Acta neuropathologica 83, 402-407.
Boya, J., Calvo, J.L., Carbonell, A.L., Borregon, A., 1991. A lectin histochemistry study on the development of rat microglial cells. Journal of anatomy 175, 229-236.
References
46
Bright, J.J., Rodriguez, M., Sriram, S., 1999. Differential influence of interleukin-12 in the pathogenesis of autoimmune and virus-induced central nervous system demyelination. Journal of virology 73, 1637-1639.
Chung, H.-S., Lee, J.-H., Kim, H., Lee, H.-J., Kim, S.-H., Kwon, H.-K., Im, S.-H., Bae, H., 2010. Foxp3 is a novel repressor of microglia activation. Glia 58, 1247-1256.
Clatch, R.J., Melvold, R.W., Dal Canto, M.C., Miller, S.D., Lipton, H.L., 1987. The Theiler's murine encephalomyelitis virus (TMEV) model for multiple sclerosis shows a strong influence of the murine equivalents of HLA-A, B, and C. Journal of neuroimmunology 15, 121-135.
Compston, A., Coles, A., 2008. Multiple sclerosis. Lancet 372, 1502-1517.
Constantinescu, C.S., Farooqi, N., O'Brien, K., Gran, B., 2011. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). British journal of pharmacology 164, 1079-1106.
D'Agostino, P.M., Gottfried-Blackmore, A., Anandasabapathy, N., Bulloch, K., 2012. Brain dendritic cells: biology and pathology. Acta neuropathologica 124, 599-614.
Dal Canto, M.C., Melvold, R.W., Kim, B.S., Miller, S.D., 1995. Two models of multiple sclerosis: experimental allergic encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV) infection. A pathological and immunological comparison. Microscopy research and technique 32, 215-229.
Dale, D.C., Boxer, L., Liles, W.C., 2008. The phagocytes: neutrophils and monocytes. Blood 112, 935-945.
David, S., Kroner, A., 2011. Repertoire of microglial and macrophage responses after spinal cord injury. Nature reviews. Neuroscience 12, 388-399.
Diemel, L.T., Copelman, C.A., Cuzner, M.L., 1998. Macrophages in CNS remyelination: friend or foe? Neurochemical research 23, 341-347.
Durafourt, B.A., Moore, C.S., Zammit, D.A., Johnson, T.A., Zaguia, F., Guiot, M.C., Bar-Or, A., Antel, J.P., 2012. Comparison of polarization properties of human adult microglia and blood-derived macrophages. Glia 60, 717-727.
Ebers, G.C., 2005. Prognostic factors for multiple sclerosis: the importance of natural history studies. Journal of neurology 252 Suppl 3, iii15-iii20.
Ensinger, E.-M., Boekhoff, T.M.A., Carlson, R., Beineke, A., Rohn, K., Tipold, A., Stein, V.M., 2010. Regional topographical differences of canine microglial immunophenotype and function in the healthy spinal cord. Journal of neuroimmunology 227, 144-152.
Fazakerley, J.K., Walker, R., 2003. Virus demyelination. Journal of neurovirology 9, 148-164.
Feuerer, M., Hill, J.A., Kretschmer, K., von Boehmer, H., Mathis, D., Benoist, C., 2010. Genomic definition of multiple ex vivo regulatory T cell subphenotypes. Proceedings of the National Academy of Sciences of the United States of America 107, 5919-5924.
References
47
Feuerer, M., Hill, J.A., Mathis, D., Benoist, C., 2009. Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nature immunology 10, 689-695.
Fletcher, J.M., Lalor, S.J., Sweeney, C.M., Tubridy, N., Mills, K.H., 2010. T cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Clinical and experimental immunology 162, 1-11.
Fox, E.J., 2004. Mechanism of action of mitoxantrone. Neurology 63, S15-18.
Fritzsching, B., Haas, J., Konig, F., Kunz, P., Fritzsching, E., Poschl, J., Krammer, P.H., Bruck, W., Suri-Payer, E., Wildemann, B., 2011. Intracerebral human regulatory T cells: analysis of CD4+ CD25+ FOXP3+ T cells in brain lesions and cerebrospinal fluid of multiple sclerosis patients. PloS one 6, e17988.
Gandhi, R., Laroni, A., Weiner, H.L., 2010. Role of the innate immune system in the pathogenesis of multiple sclerosis. Journal of neuroimmunology 221, 7-14.
Gao, Z., Tsirka, S.E., 2011. Animal Models of MS Reveal Multiple Roles of Microglia in Disease Pathogenesis. Neurology research international 2011, 383087.
Gay, F.W., Drye, T.J., Dick, G.W., Esiri, M.M., 1997. The application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis. Identification and characterization of the primary demyelinating lesion. Brain : a journal of neurology 120 ( Pt 8), 1461-1483.
Gerhauser, I., Hansmann, F., Puff, C., Kumnok, J., Schaudien, D., Wewetzer, K., Baumgärtner, W., 2012. Theiler's murine encephalomyelitis virus induced phenotype switch of microglia in vitro. Journal of neuroimmunology 252, 49-55.
Giunti, D., Parodi, B., Cordano, C., Uccelli, A., Kerlero de Rosbo, N., 2014. Can we switch microglia's phenotype to foster neuroprotection? Focus on multiple sclerosis. Immunology 141, 328-339.
Gobel, K., Bittner, S., Melzer, N., Pankratz, S., Dreykluft, A., Schuhmann, M.K., Meuth, S.G., Wiendl, H., 2012. CD4(+) CD25(+) FoxP3(+) regulatory T cells suppress cytotoxicity of CD8(+) effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level. J Neuroinflammation 9, 41.
Goldmann, T., Prinz, M., 2013. Role of microglia in CNS autoimmunity. Clinical & developmental immunology 2013, 208093.
Gordon, S., Martinez, F.O., 2010. Alternative activation of macrophages: mechanism and functions. Immunity 32, 593-604.
Griot, C., Vandevelde, M., Richard, A., Peterhans, E., Stocker, R., 1990. Selective degeneration of oligodendrocytes mediated by reactive oxygen species. Free radical research communications 11, 181-193.
Gudi, V., Moharregh-Khiabani, D., Skripuletz, T., Koutsoudaki, P.N., Kotsiari, A., Skuljec, J., Trebst, C., Stangel, M., 2009. Regional differences between grey and white matter in cuprizone induced demyelination. Brain research 1283, 127-138.
Gupta, N., Hegde, P., Lecerf, M., Nain, M., Kaur, M., Kalia, M., Vrati, S., Bayry, J., Lacroix-Desmazes, S., Kaveri, S.V., 2014. Japanese encephalitis virus expands regulatory T cells by increasing the expression of PD-L1 on dendritic cells. European journal of immunology 44, 1363-1374.
Hall, G.L., Wing, M.G., Compston, D.A., Scolding, N.J., 1997. beta-Interferon regulates the immunomodulatory activity of neonatal rodent microglia. Journal of neuroimmunology 72, 11-19.
References
48
Hamilton, S.P., Rome, L.H., 1994. Stimulation of in vitro myelin synthesis by microglia. Glia 11, 326-335.
Haroon, F., Drogemuller, K., Handel, U., Brunn, A., Reinhold, D., Nishanth, G., Mueller, W., Trautwein, C., Ernst, M., Deckert, M., Schluter, D., 2011. Gp130-dependent astrocytic survival is critical for the control of autoimmune central nervous system inflammation. Journal of immunology 186, 6521-6531.
Hendriks, J.J., Teunissen, C.E., de Vries, H.E., Dijkstra, C.D., 2005. Macrophages and neurodegeneration. Brain research. Brain research reviews 48, 185-195.
Herder, V., Gerhauser, I., Klein, S.K., Almeida, P., Kummerfeld, M., Ulrich, R., Seehusen, F., Rohn, K., Schaudien, D., Baumgärtner, W., Huehn, J., Beineke, A., 2012a. Interleukin-10 expression during the acute phase is a putative prerequisite for delayed viral elimination in a murine model for multiple sclerosis. Journal of neuroimmunology 249, 27-39.
Herder, V., Hansmann, F., Stangel, M., Schaudien, D., Rohn, K., Baumgärtner, W., Beineke, A., 2012b. Cuprizone inhibits demyelinating leukomyelitis by reducing immune responses without virus exacerbation in an infectious model of multiple sclerosis. Journal of neuroimmunology 244, 84-93.
Horikoshi, Y., Sasaki, A., Taguchi, N., Maeda, M., Tsukagoshi, H., Sato, K., Yamaguchi, H., 2003. Human GLUT5 immunolabeling is useful for evaluating microglial status in neuropathological study using paraffin sections. Acta neuropathologica 105, 157-162.
Hou, S.W., Liu, C.Y., Li, Y.H., Yu, J.Z., Feng, L., Liu, Y.T., Guo, M.F., Xie, Y., Meng, J., Zhang, H.F., Xiao, B.G., Ma, C.G., 2012. Fasudil ameliorates disease progression in experimental autoimmune encephalomyelitis, acting possibly through antiinflammatory effect. CNS neuroscience & therapeutics 18, 909-917.
Huang, X., Stone, D.K., Yu, F., Zeng, Y., Gendelman, H.E., 2010. Functional proteomic analysis for regulatory T cell surveillance of the HIV-1-infected macrophage. Journal of proteome research 9, 6759-6773.
Hughes, J.E., Srinivasan, S., Lynch, K.R., Proia, R.L., Ferdek, P., Hedrick, C.C., 2008. Sphingosine-1-phosphate induces an antiinflammatory phenotype in macrophages. Circulation research 102, 950-958.
Iarlori, C., Gambi, D., Lugaresi, A., Patruno, A., Felaco, M., Salvatore, M., Speranza, L., Reale, M., 2008. Reduction of free radicals in multiple sclerosis: effect of glatiramer acetate (Copaxone). Multiple sclerosis 14, 739-748.
Ito, D., Imai, Y., Ohsawa, K., Nakajima, K., Fukuuchi, Y., Kohsaka, S., 1998. Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain research. Molecular brain research 57, 1-9.
Jee, Y., Piao, W.H., Liu, R., Bai, X.F., Rhodes, S., Rodebaugh, R., Campagnolo, D.I., Shi, F.D., Vollmer, T.L., 2007. CD4(+)CD25(+) regulatory T cells contribute to the therapeutic effects of glatiramer acetate in experimental autoimmune encephalomyelitis. Clinical immunology 125, 34-42.
Johnson, K.P., Brooks, B.R., Cohen, J.A., Ford, C.C., Goldstein, J., Lisak, R.P., Myers, L.W., Panitch, H.S., Rose, J.W., Schiffer, R.B., 1995. Copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: results of a phase III multicenter, double-blind placebo-controlled trial. The Copolymer 1 Multiple Sclerosis Study Group. Neurology 45, 1268-1276.
Jung, S., Siglienti, I., Grauer, O., Magnus, T., Scarlato, G., Toyka, K., 2004. Induction of IL-10 in rat peritoneal macrophages and dendritic cells by glatiramer acetate. Journal of neuroimmunology 148, 63-7
References
49
Kennedy, K.J., Strieter, R.M., Kunkel, S.L., Lukacs, N.W., Karpus, W.J., 1998. Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1α and monocyte chemotactic protein-1. Journal of neuroimmunology 92, 98-108.
Kettenmann, H., Hanisch, U.K., Noda, M., Verkhratsky, A., 2011. Physiology of microglia. Physiological reviews 91, 461-553.
Kigerl, K.A., Gensel, J.C., Ankeny, D.P., Alexander, J.K., Donnelly, D.J., Popovich, P.G., 2009. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. The Journal of neuroscience : the official journal of the Society for Neuroscience 29, 13435-13444.
Kim, B., Palma, J., Kwon, D., Fuller, A., 2005a. Innate immune response induced by Theiler's murine encephalomyelitis virus infection. Immunol Res 31, 1-12.
Kim, B.S., Lyman, M.A., Kang, B.S., Kang, H.K., Lee, H.G., Mohindru, M., Palma, J.P., 2001. Pathogenesis of virus-induced immune-mediated demyelination. Immunol Res 24, 121-130.
Kim, Y.S., Kim, S.S., Cho, J.J., Choi, D.H., Hwang, O., Shin, D.H., Chun, H.S., Beal, M.F., Joh, T.H., 2005b. Matrix metalloproteinase-3: a novel signaling proteinase from apoptotic neuronal cells that activates microglia. The Journal of neuroscience : the official journal of the Society for Neuroscience 25, 3701-3711.
Kobayashi, K., Imagama, S., Ohgomori, T., Hirano, K., Uchimura, K., Sakamoto, K., Hirakawa, A., Takeuchi, H., Suzumura, A., Ishiguro, N., Kadomatsu, K., 2013. Minocycline selectively inhibits M1 polarization of microglia. Cell death & disease 4, e525.
Kopadze, T., Dehmel, T., Hartung, H.P., Stuve, O., Kieseier, B.C., 2006. Inhibition by mitoxantrone of in vitro migration of immunocompetent cells: a possible mechanism for therapeutic efficacy in the treatment of multiple sclerosis. Archives of neurology 63, 1572-1578.
Korn, T., Anderson, A.C., Bettelli, E., Oukka, M., 2007. The dynamics of effector T cells and Foxp3+ regulatory T cells in the promotion and regulation of autoimmune encephalomyelitis. Journal of neuroimmunology 191, 51-60.
Kuerten, S., Kostova-Bales, D.A., Frenzel, L.P., Tigno, J.T., Tary-Lehmann, M., Angelov, D.N., Lehmann, P.V., 2007. MP4- and MOG:35-55-induced EAE in C57BL/6 mice differentially targets brain, spinal cord and cerebellum. Journal of neuroimmunology 189, 31-40.
Kummerfeld, M., Meens, J., Haas, L., Baumgärtner, W., Beineke, A., 2009. Generation and characterization of a polyclonal antibody for the detection of Theiler's murine encephalomyelitis virus by light and electron microscopy. Journal of virological methods 160, 185-188.
Kummerfeld, M., Seehusen, F., Klein, S., Ulrich, R., Kreutzer, R., Gerhauser, I., Herder, V., Baumgärtner, W., Beineke, A., 2012. Periventricular demyelination and axonal pathology is associated with subependymal virus spread in a murine model for multiple sclerosis. Intervirology 55, 401-416.
Kurtzke, J.F., 1993. Epidemiologic evidence for multiple sclerosis as an infection. Clinical microbiology reviews 6, 382-427.
Laskin, D.L., 2009. Macrophages and inflammatory mediators in chemical toxicity: a battle of forces. Chemical research in toxicology 22, 1376-1385.
References
50
Lassmann, H., Bruck, W., Lucchinetti, C., 2001. Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy. Trends in molecular medicine 7, 115-121.
Lassmann, H., Rinner, W., Hickey, W.F., 1994. Differential role of hematogenous macrophages, resident microglia and astrocytes in antigen presentation and tissue damage during autoimmune encephalomyelitis. Neuropathology and applied neurobiology 20, 195-196.
Lassmann, H., van Horssen, J., 2011. The molecular basis of neurodegeneration in multiple sclerosis. FEBS letters 585, 3715-3723.
Li, W.W., Setzu, A., Zhao, C., Franklin, R.J., 2005. Minocycline-mediated inhibition of microglia activation impairs oligodendrocyte progenitor cell responses and remyelination in a non-immune model of demyelination. Journal of neuroimmunology 158, 58-66.
Liesz, A., Suri-Payer, E., Veltkamp, C., Doerr, H., Sommer, C., Rivest, S., Giese, T., Veltkamp, R., 2009. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med 15, 192-199.
Lipton, H.L., Melvold, R., 1984. Genetic analysis of susceptibility to Theiler's virus-induced demyelinating disease in mice. Journal of immunology 132, 1821-1825.
Lipton, H.L., Melvold, R., Miller, S.D., Dal Canto, M.C., 1995. Mutation of a major histocompatibility class I locus, H-2D, leads to an increased virus burden and disease susceptibility in Theiler's virus-induced demyelinating disease. Journal of neurovirology 1, 138-144.
Liu, C., Li, Y., Yu, J., Feng, L., Hou, S., Liu, Y., Guo, M., Xie, Y., Meng, J., Zhang, H., Xiao, B., Ma, C., 2013. Targeting the shift from M1 to M2 macrophages in experimental autoimmune encephalomyelitis mice treated with fasudil. PloS one 8, e54841.
Liu, J., Gong, N., Huang, X., Reynolds, A.D., Mosley, R.L., Gendelman, H.E., 2009. Neuromodulatory activities of CD4+CD25+ regulatory T cells in a murine model of HIV-1-associated neurodegeneration. Journal of immunology 182, 3855-3865.
Liu, Y., Teige, I., Birnir, B., Issazadeh-Navikas, S., 2006. Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nature medicine 12, 518-525.
Liuzzi, G.M., Riccio, P., Dal Canto, M.C., 1995a. Release of myelin basic protein-degrading proteolytic activity from microglia and macrophages after infection with Theiler's murine encephalomyelitis virus: comparison between susceptible and resistant mice. Journal of neuroimmunology 62, 91-102.
Liuzzi, G.M., Riccio, P., Dal Canto, M.C., 1995b. Release of myelin basic protein-degrading proteolytic activity from microglia and macrophages after infection with Theiler's murine encephalomyelitis virus: comparison between susceptible and resistant mice. Journal of neuroimmunology 62, 91-102.
Loughlin, A.J., Copelman, C.A., Hall, A., Armer, T., Young, B.C., Landon, D.N., Cuzner, M.L., 1997. Myelination and remyelination of aggregate rat brain cell cultures enriched with macrophages. Journal of neuroscience research 47, 384-392.
Lowther, D.E., Hafler, D.A., 2012. Regulatory T cells in the central nervous system. Immunological reviews 248, 156-169.
Lucchinetti, C., Bruck, W., Parisi, J., Scheithauer, B., Rodriguez, M., Lassmann, H., 2000. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Annals of neurology 47, 707-717.
References
51
Lund, J.M., Hsing, L., Pham, T.T., Rudensky, A.Y., 2008. Coordination of early protective immunity to viral infection by regulatory T cells. Science 320, 1220-1224.
Ma, L., Morton, A.J., Nicholson, L.F., 2003. Microglia densitiy decreases with age in a mouse model of Huntington's disease. Glia 43, 274-280.
MacDonald, A.J., Duffy, M., Brady, M.T., McKiernan, S., Hall, W., Hegarty, J., Curry, M., Mills, K.H., 2002. CD4 T helper type 1 and regulatory T cells induced against the same epitopes on the core protein in hepatitis C virus-infected persons. The Journal of infectious diseases 185, 720-727.
Mackaness, G.B., 1977. Cellular immunity and the parasite. Advances in experimental medicine and biology 93, 65-73.
Maher, F., Vannucci, S.J., Simpson, I.A., 1994. Glucose transporter proteins in brain. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 8, 1003-1011.
Mantovani, A., Sozzani, S., Locati, M., Schioppa, T., Saccani, A., Allavena, P., Sica, A., 2004. Infiltration of tumours by macrophages and dendritic cells: tumour-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Novartis Foundation symposium 256, 137-145; discussion 146-138, 259-169.
Marik, C., Felts, P.A., Bauer, J., Lassmann, H., Smith, K.J., 2007. Lesion genesis in a subset of patients with multiple sclerosis: a role for innate immunity? Brain : a journal of neurology 130, 2800-2815.
Martinez, F.O., Sica, A., Mantovani, A., Locati, M., 2008. Macrophage activation and polarization. Frontiers in bioscience : a journal and virtual library 13, 453-461.
Martinez, N.E., Karlsson, F., Sato, F., Kawai, E., Omura, S., Minagar, A., Grisham, M.B., Tsunoda, I., 2014. Protective and Detrimental Roles for Regulatory T Cells in a Viral Model for Multiple Sclerosis. Brain pathology.
Mattei, D., Djodari-Irani, A., Hadar, R., Pelz, A., de Cossio, L.F., Goetz, T., Matyash, M., Kettenmann, H., Winter, C., Wolf, S.A., 2014. Minocycline rescues decrease in neurogenesis, increase in microglia cytokines and deficits in sensorimotor gating in an animal model of schizophrenia. Brain, behavior, and immunity 38, 175-184.
McMahon, E.J., Bailey, S.L., Castenada, C.V., Waldner, H., Miller, S.D., 2005. Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nature medicine 11, 335-339.
Mecha, M., Carrillo-Salinas, F.J., Mestre, L., Feliu, A., Guaza, C., 2013. Viral models of multiple sclerosis: neurodegeneration and demyelination in mice infected with Theiler's virus. Progress in neurobiology 101-102, 46-64.
Merrill, J.E., Ignarro, L.J., Sherman, M.P., Melinek, J., Lane, T.E., 1993. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. Journal of immunology 151, 2132-2141.
Mikita, J., Dubourdieu-Cassagno, N., Deloire, M.S., Vekris, A., Biran, M., Raffard, G., Brochet, B., Canron, M.H., Franconi, J.M., Boiziau, C., Petry, K.G., 2011. Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis. Amelioration of clinical status by M2 activated monocyte administration. Multiple sclerosis 17, 2-15.
Minagar, A., Zivadinov, R., 2011. Pathophysiology of demyelinating disorders. Pathophysiology : the official journal of the International Society for Pathophysiology / ISP 18, 1-2.
References
52
Miron, V.E., Boyd, A., Zhao, J.W., Yuen, T.J., Ruckh, J.M., Shadrach, J.L., van Wijngaarden, P., Wagers, A.J., Williams, A., Franklin, R.J., ffrench-Constant, C., 2013. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nature neuroscience 16, 1211-1218.
Miron, V.E., Franklin, R.J., 2014. Macrophages and CNS remyelination. Journal of neurochemistry.
Nandakumar, S., Miller, C.W., Kumaraguru, U., 2009. T regulatory cells: an overview and intervention techniques to modulate allergy outcome. Clinical and molecular allergy : CMA 7, 5.
Nataf, S., 2009. Neuroinflammation responses and neurodegeneration in multiple sclerosis. Revue neurologique 165, 1023-1028.
Nikolakopoulou, A.M., Dutta, R., Chen, Z., Miller, R.H., Trapp, B.D., 2013. Activated microglia enhance neurogenesis via trypsinogen secretion. Proceedings of the National Academy of Sciences of the United States of America 110, 8714-8719.
O'Shea, J.J., Hunter, C.A., Germain, R.N., 2008. T cell heterogeneity: firmly fixed, predominantly plastic or merely malleable? Nature immunology 9, 450-453.
Oleszak, E.L., Chang, J.R., Friedman, H., Katsetos, C.D., Platsoucas, C.D., 2004. Theiler's virus infection: a model for multiple sclerosis. Clinical microbiology reviews 17, 174-207.
Olson, J.K., 2010. Immune response by microglia in the spinal cord. Annals of the New York Academy of Sciences 1198, 271-278.
Olson, J.K., Girvin, A.M., Miller, S.D., 2001. Direct activation of innate and antigen-presenting functions of microglia following infection with Theiler's virus. Journal of virology 75, 9780-9789.
Palma, J.P., Kim, B.S., 2004. The scope and activation mechanisms of chemokine gene expression in primary astrocytes following infection with Theiler's virus. Journal of neuroimmunology 149, 121-129.
Pinkston, J.B., Kablinger, A., Alekseeva, N., 2007. Multiple Sclerosis and Behavior, In: International review of neurobiology. Academic Press, pp. 323-339.
Pringproa, K., Rohn, K., Kummerfeld, M., Wewetzer, K., Baumgärtner, W., 2010. Theiler's murine encephalomyelitis virus preferentially infects immature stages of the murine oligodendrocyte precursor cell line BO-1 and blocks oligodendrocytic differentiation in vitro. Brain research 1327, 24-37.
Rawji, K.S., Yong, V.W., 2013. The benefits and detriments of macrophages/microglia in models of multiple sclerosis. Clinical & developmental immunology 2013, 948976.
Richards, M.H., Getts, M.T., Podojil, J.R., Jin, Y.H., Kim, B.S., Miller, S.D., 2011. Virus expanded regulatory T cells control disease severity in the Theiler's virus mouse model of MS. Journal of autoimmunity 36, 142-154.
Rock, R.B., Gekker, G., Hu, S., Sheng, W.S., Cheeran, M., Lokensgard, J.R., Peterson, P.K., 2004. Role of microglia in central nervous system infections. Clinical microbiology reviews 17, 942-964, table of contents.
Rosati, G., 2001. The prevalence of multiple sclerosis in the world: an update. Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 22, 117-139.
References
53
Rosen, H., Goetzl, E.J., 2005. Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network. Nature reviews. Immunology 5, 560-570.
Rossi, C.P., Delcroix, M., Huitinga, I., McAllister, A., van Rooijen, N., Claassen, E., Brahic, M., 1997. Role of macrophages during Theiler's virus infection. Journal of virology 71, 3336-3340.
Roussarie, J.P., Ruffie, C., Brahic, M., 2007. The role of myelin in Theiler's virus persistence in the central nervous system. PLoS pathogens 3, e23.
Saijo, K., Glass, C.K., 2011. Microglial cell origin and phenotypes in health and disease. Nature reviews. Immunology 11, 775-787.
Sakaguchi, S., 2003. Regulatory T cells: mediating compromises between host and parasite. Nat Immunol 4, 10-11.
Sakaguchi, S., Ono, M., Setoguchi, R., Yagi, H., Hori, S., Fehervari, Z., Shimizu, J., Takahashi, T., Nomura, T., 2006. Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunological reviews 212, 8-27.
Sato, S., Reiner, S.L., Jensen, M.A., Roos, R.P., 1997. Central nervous system cytokine mRNA expression following Theiler's murine encephalomyelitis virus infection. Journal of neuroimmunology 76, 213-223.
Schilling, S., Goelz, S., Linker, R., Luehder, F., Gold, R., 2006. Fumaric acid esters are effective in chronic experimental autoimmune encephalomyelitis and suppress macrophage infiltration. Clinical and experimental immunology 145, 101-107.
Skripuletz, T., Lindner, M., Kotsiari, A., Garde, N., Fokuhl, J., Linsmeier, F., Trebst, C., Stangel, M., 2008. Cortical Demyelination Is Prominent in the Murine Cuprizone Model and Is Strain-Dependent. The American journal of pathology 172, 1053-1061.
Skripuletz, T., Miller, E., Moharregh-Khiabani, D., Blank, A., Pul, R., Gudi, V., Trebst, C., Stangel, M., 2010. Beneficial effects of minocycline on cuprizone induced cortical demyelination. Neurochemical research 35, 1422-1433.
Spitzbarth, I., Bock, P., Haist, V., Stein, V.M., Tipold, A., Wewetzer, K., Baumgärtner, W., Beineke, A., 2011. Prominent microglial activation in the early proinflammatory immune response in naturally occurring canine spinal cord injury. Journal of neuropathology and experimental neurology 70, 703-714.
Steffen, B.J., Butcher, E.C., Engelhardt, B., 1994. Evidence for involvement of ICAM-1 and VCAM-1 in lymphocyte interaction with endothelium in experimental autoimmune encephalomyelitis in the central nervous system in the SJL/J mouse. The American journal of pathology 145, 189-201.
Stein, V.M., Czub, M., Schreiner, N., Moore, P.F., Vandevelde, M., Zurbriggen, A., Tipold, A., 2004. Microglial cell activation in demyelinating canine distemper lesions. Journal of neuroimmunology 153, 122-131.
Stein, V.M., Schreiner, N.M.S., Moore, P.F., Vandevelde, M., Zurbriggen, A., Tipold, A., 2008. Immunophenotypical characterization of monocytes in canine distemper virus infection. Veterinary microbiology 131, 237-246.
Streit, W.J., Kreutzberg, G.W., 1987. Lectin binding by resting and reactive microglia. Journal of neurocytology 16, 249-260.
References
54
t Hart, B.A., Gran, B., Weissert, R., 2011. EAE: imperfect but useful models of multiple sclerosis. Trends in molecular medicine 17, 119-125.
Thomas, W.E., 1999. Brain macrophages: on the role of pericytes and perivascular cells. Brain research. Brain research reviews 31, 42-57.
Thompson, A.J., Toosy, A.T., Ciccarelli, O., 2010. Pharmacological management of symptoms in multiple sclerosis: current approaches and future directions. Lancet neurology 9, 1182-1199.
Tiemessen, M.M., Jagger, A.L., Evans, H.G., van Herwijnen, M.J., John, S., Taams, L.S., 2007. CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages. Proceedings of the National Academy of Sciences of the United States of America 104, 19446-19451.
Trajkovic, V., Vuckovic, O., Stosic-Grujicic, S., Miljkovic, D., Popadic, D., Markovic, M., Bumbasirevic, V., Backovic, A., Cvetkovic, I., Harhaji, L., Ramic, Z., Mostarica Stojkovic, M., 2004. Astrocyte-induced regulatory T cells mitigate CNS autoimmunity. Glia 47, 168-179.
Trojano, M., Liguori, M., Paolicelli, D., Zimatore, G.B., De Robertis, F., Avolio, C., Giuliani, F., Fuiani, A., Livrea, P., Southern Italy, M.S.G., 2003. Interferon beta in relapsing-remitting multiple sclerosis: an independent postmarketing study in southern Italy. Multiple sclerosis 9, 451-457.
Ulrich, R., Kalkuhl, A., Deschl, U., Baumgärtner, W., 2010. Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis. Journal of cellular and molecular medicine 14, 434-448.
Ulrich, R., Seeliger, F., Kreutzer, M., Germann, P.G., Baumgärtner, W., 2008. Limited remyelination in Theiler's murine encephalomyelitis due to insufficient oligodendroglial differentiation of nerve/glial antigen 2 (NG2)-positive putative oligodendroglial progenitor cells. Neuropathology and applied neurobiology 34, 603-620.
van der Valk, P., De Groot, C.J., 2000. Staging of multiple sclerosis (MS) lesions: pathology of the time frame of MS. Neuropathology and applied neurobiology 26, 2-10.
van Horssen, J., Witte, M.E., Schreibelt, G., de Vries, H.E., 2011. Radical changes in multiple sclerosis pathogenesis. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1812, 141-150.
Vannucci, S.J., Maher, F., Simpson, I.A., 1997. Glucose transporter proteins in brain: delivery of glucose to neurons and glia. Glia 21, 2-21.
Vereyken, E.J., Heijnen, P.D., Baron, W., de Vries, E.H., Dijkstra, C.D., Teunissen, C.E., 2011. Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types. Journal of neuroinflammation 8, 58.
Vignali, D.A., Collison, L.W., Workman, C.J., 2008. How regulatory T cells work. Nature reviews. Immunology 8, 523-532.
Vollmer, T., Stewart, T., Baxter, N., 2010. Mitoxantrone and cytotoxic drugs' mechanisms of action. Neurology 74 Suppl 1, S41-46.
Wainwright, D.A., Balyasnikova, I.V., Chang, A.L., Ahmed, A.U., Moon, K.S., Auffinger, B., Tobias, A.L., Han, Y., Lesniak, M.S., 2012. IDO expression in brain tumors increases the recruitment of regulatory T cells and negatively impacts survival. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 6110-6121.
References
55
Wainwright, D.A., Chang, A.L., Dey, M., Balyasnikova, I.V., Kim, C., Tobias, A.L., Cheng, Y., Kim, J., Zhang, L., Qiao, J., Han, Y., Lesniak, M.S., 2014. Durable therapeutic efficacy utilizing combinatorial blockade against IDO, CTLA-4 and PD-L1 in mice with brain tumors. Clinical cancer research : an official journal of the American Association for Cancer Research.
Wang, X., Haroon, F., Karray, S., Martina, D., Schlüter, D., 2013. Astrocytic Fas ligand expression is required to induce T-cell apoptosis and recovery from experimental autoimmune encephalomyelitis. European journal of immunology 43, 115-124.
Watson, C.M., Davison, A.N., Baker, D., O'Neill, J.K., Turk, J.L., 1991. Suppression of demyelination by mitoxantrone. International journal of immunopharmacology 13, 923-930.
Weber, M.S., Starck, M., Wagenpfeil, S., Meinl, E., Hohlfeld, R., Farina, C., 2004. Multiple sclerosis: glatiramer acetate inhibits monocyte reactivity in vitro and in vivo. Brain : a journal of neurology 127, 1370-1378.
Wierinckx, A., Brevé, J., Mercier, D., Schultzberg, M., Drukarch, B., Van Dam, A.-M., 2005. Detoxication enzyme inducers modify cytokine production in rat mixed glial cells. Journal of neuroimmunology 166, 132-
Zajicek, J.P., Wing, M., Scolding, N.J., Compston, D.A., 1992. Interactions between oligodendrocytes and microglia. A major role for complement and tumour necrosis factor in oligodendrocyte adherence and killing. Brain : a journal of neurology 115 ( Pt 6), 1611-1631.
Zhang, G.X., Li, J., Ventura, E., Rostami, A., 2002. Parenchymal microglia of naive adult C57BL/6J mice express high levels of B7.1, B7.2, and MHC class II. Experimental and molecular pathology 73, 35-45.
Zhang, Z., Zhang, Z.Y., Schluesener, H.J., 2009. Compound A, a plant origin ligand of glucocorticoid receptors, increases regulatory T cells and M2 macrophages to attenuate experimental autoimmune neuritis with reduced side effects. Journal of immunology 183, 3081-3091.
Zhao, J., Zhao, J., Fett, C., Trandem, K., Fleming, E., Perlman, S., 2011. IFN-gamma- and IL-10-expressing virus epitope-specific Foxp3(+) T reg cells in the central nervous system during encephalomyelitis. The Journal of experimental medicine 208, 1571-1577.
Zhao, W., Beers, D.R., Liao, B., Henkel, J.S., Appel, S.H., 2012. Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms. Neurobiology of disease 48, 418-428.
Zoecklein, L.J., Pavelko, K.D., Gamez, J., Papke, L., McGavern, D.B., Ure, D.R., Njenga, M.K., Johnson, A.J., Nakane, S., Rodriguez, M., 2003. Direct Comparison of Demyelinating Disease Induced by the Daniel's Strain and BeAn Strain of Theiler's Murine Encephalomyelitis Virus. Brain pathology 13, 291-308.
Zozulya, A.L., Ortler, S., Lee, J., Weidenfeller, C., Sandor, M., Wiendl, H., Fabry, Z., 2009. Intracerebral dendritic cells critically modulate encephalitogenic versus regulatory immune responses in the CNS. The Journal of neuroscience : the official journal of the Society for Neuroscience 29, 140-152.
Attachments
56
9. ATTACHMENTS
9.1 Supplemental material to chapter 2
Table 1: Correlation between data obtained by gene expression analyses and immunofluorescence
Immunofluorescence Gene expression CD107b CD68 CD16/32 Arginase-1
Arginase-1 0.756* 0.686* 0.650* 0.630* Cd68 0.735* 0.751* 0.757* 0.708* Cd32b 0.722* 0.854* 0.760* 0.624* Cd16 0.723* 0.852* 0.804* 0.589* Cd107b 0.629* 0.629* 0.636* 0.476* Spearman’s rank correlation coefficient was used to correlate absolute numbers (positive cells/spinal cord) of CD107b+, CD68+, CD16/32+, and aginase-1+ cells with the respective mRNA level measured by microarray analysis in the spinal cord of Theiler´s murine encephalomyelitis virus-infected mice. Significant differences of the correlation coefficient from zero are marked as follows: * = p≤0.01.
Attachments
57
Table S1: list of selected M1- and M2-related genes
Probesets Gene symbol Gene title Polarity
1416006_at Mdk midkine M1
1416069_at Pfkp phosphofructokinase, platelet M1
1416432_at Pfkfb3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 M1
1416654_at Slc31a2 solute carrier family 31, member 2 M1
1417189_at Psme2 proteasome (prosome, macropain) 28 subunit, beta M1
1417190_at Nampt nicotinamide phosphoribosyltransferase M1
1417244_a_at Irf7 interferon regulatory factor 7 M1
1417785_at Pla1a phospholipase A1 member A M1
1417876_at Fcgr1 Fc receptor, IgG, high affinity I M1
1418126_at Ccl5 chemokine (C-C motif) ligand 5 M1
1418219_at Il15 interleukin 15 M1
1418326_at Slc7a5 solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 M1
1418652_at Cxcl9 chemokine (C-X-C motif) ligand 9 M1
1418666_at Ptx3 pentraxin related gene M1
1418930_at Cxcl10 chemokine (C-X-C motif) ligand 10 M1
1419004_s_at Bcl2a1a B-cell leukemia/lymphoma 2 related protein A1a /// B-cell leukemia/lymphoma 2 related protein A1b /// B-cell leukemia/lymphoma 2 related protein A1d M1
1419282_at Ccl12 chemokine (C-C motif) ligand 12 M1
1419529_at Il23a interleukin 23, alpha subunit p19 M1
1419536_a_at Rela v-rel reticuloendotheliosis viral oncogene homolog A (avian) M1
1419561_at Ccl3 chemokine (C-C motif) ligand 3 M1
1419684_at Ccl8 chemokine (C-C motif) ligand 8 M1
1419697_at Cxcl11 chemokine (C-X-C motif) ligand 11 M1
1420393_at Nos2 nitric oxide synthase 2, inducible M1
1420437_at Ido1 indoleamine 2,3-dioxygenase 1 M1
1420604_at Hesx1 homeobox gene expressed in ES cells M1
1420692_at Il2ra interleukin 2 receptor, alpha chain M1
1421228_at Ccl7 chemokine (C-C motif) ligand 7 M1
1421392_a_at Birc3 baculoviral IAP repeat-containing 3 M1
1421578_at Ccl4 chemokine (C-C motif) ligand 4 M1
1421812_at Tapbp TAP binding protein M1
1422957_at Ccr3 chemokine (C-C motif) receptor 3 M1
1423466_at Ccr7 chemokine (C-C motif) receptor 7 M1
1423756_s_at Igfbp4 insulin-like growth factor binding protein 4 M1
1423760_at Cd44 CD44 antigen M1
1424067_at Icam1 intercellular adhesion molecule 1 M1
1424339_at Oasl1 2'-5' oligoadenylate synthetase-like 1 M1
1425065_at Oas2 2'-5' oligoadenylate synthetase 2 M1
1425137_a_at H2-Q10 histocompatibility 2, Q region locus 10 M1
1425454_a_at Il12a interleukin 12a M1
Attachments
58
Table S1: list of selected M1- and M2-related genes (continued)
Probesets Gene symbol Gene title Polarity
1425947_at Ifng interferon gamma M1
1427256_at Vcan versican M1
1427705_a_at Nfkb1 nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, p105 M1
1427717_at Cd80 CD80 antigen M1
1429195_at Apol6 apolipoprotein L 6 M1
1432436_a_at Ak3 adenylate kinase 3 M1
1434015_at Slc2a6 solute carrier family 2 (facilitated glucose transporter), member 6 M1
1435477_s_at Fcgr2b Fc receptor, IgG, low affinity IIb M1
1438148_at Cxcl3 chemokine (C-X-C motif) ligand 3 M1
1439221_s_at Cd40 CD40 antigen M1
1439588_at Slco5a1 solute carrier organic anion transporter family, member 5A1 M1
1439680_at Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10 M1
1441054_at Apol8 apolipoprotein L 8 M1
1448206_at Psma2 proteasome (prosome, macropain) subunit, alpha type 2 M1
1448436_a_at Irf1 interferon regulatory factor 1 M1
1448575_at Il7r interleukin 7 receptor M1
1448620_at Fcgr3 Fc receptor, IgG, low affinity III M1
1448681_at Il15ra interleukin 15 receptor, alpha chain M1
1448823_at Cxcl12 chemokine (C-X-C motif) ligand 12 M1
1448862_at Icam2 intercellular adhesion molecule 2 M1
1449025_at Ifit3 interferon-induced protein with tetratricopeptide repeats 3 M1
1449038_at Hsd11b1 hydroxysteroid 11-beta dehydrogenase 1 M1
1449049_at Tlr1 toll-like receptor 1 M1
1449277_at Ccl19 chemokine (C-C motif) ligand 19 M1
1449363_at Atf3 activating transcription factor 3 M1
1449497_at Il12b interleukin 12b M1
1450696_at Psmb9 proteasome (prosome, macropain) subunit, beta type 9 (large multifunctional peptidase 2) M1
1451318_a_at Lyn Yamaguchi sarcoma viral (v-yes-1) oncogene homolog M1
1451924_a_at Edn1 endothelin 1 M1
1453851_a_at Gadd45g growth arrest and DNA-damage-inducible 45 gamma M1
1453913_a_at Tap2 transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) M1
1458291_at Inhba Inhibin beta-A M1
1460251_at Fas Fas (TNF receptor superfamily member 6) M1
1417262_at Ptgs2 prostaglandin-endoperoxide synthase 2 M1, M2
1417851_at Cxcl13 chemokine (C-X-C motif) ligand 13 M1, M2
1418711_at Pdgfa platelet derived growth factor, alpha M1, M2
1419132_at Tlr2 toll-like receptor 2 M1, M2
1419209_at Cxcl1 chemokine (C-X-C motif) ligand 1 M1, M2
1419607_at Tnf tumor necrosis factor M1, M2
Attachments
59
Table S1: list of selected M1- and M2-related genes (continued)
Probesets Gene symbol Gene title Polarity
1421473_at Il1a interleukin 1 alpha M1, M2
1422029_at Ccl20 chemokine (C-C motif) ligand 20 M1, M2
1449399_a_at Il1b interleukin 1 beta M1, M2
1449858_at Cd86 CD86 antigen M1, M2
1450297_at Il6 interleukin 6 M1, M2
1451596_a_at Sphk1 sphingosine kinase 1 M1, M2
1452431_s_at H2-Aa histocompatibility 2, class II antigen A, alpha M1, M2
1416086_at Tpst2 protein-tyrosine sulfotransferase 2 M2
1416382_at Ctsc cathepsin C M2
1417268_at Cd14 CD14 antigen M2
1417391_a_at Il16 interleukin 16 M2
1417597_at Cd28 CD28 antigen M2
1417702_a_at Hnmt histamine N-methyltransferase M2
1417859_at Gas7 growth arrest specific 7 M2
1417925_at Ccl22 chemokine (C-C motif) ligand 22 M2
1418340_at Fcer1g Fc receptor, IgE, high affinity I, gamma polypeptide M2
1418826_at Ms4a6b membrane-spanning 4-domains, subfamily A, member 6B M2
1418982_at Cebpa CCAAT/enhancer binding protein (C/EBP), alpha M2
1419144_at Cd163 CD163 antigen M2
1419210_at Hrh1 histamine receptor H1 M2
1419413_at Ccl17 chemokine (C-C motif) ligand 17 M2
1419549_at Arg1 arginase, liver M2
1419696_at Cd4 CD4 antigen M2
1419764_at Chi3l3 chitinase 3-like 3 M2
1419873_s_at Csf1r colony stimulating factor 1 receptor M2
1420338_at Alox15 arachidonate 15-lipoxygenase M2
1420380_at Ccl2 chemokine (C-C motif) ligand 2 M2
1420653_at Tgfb1 transforming growth factor, beta 1 M2
1420699_at Clec7a C-type lectin domain family 7, member a M2
1421688_a_at Ccl1 chemokine (C-C motif) ligand 1 M2
1421775_at Fcer1a Fc receptor, IgE, high affinity I, alpha polypeptide M2
1421855_at Fgl2 fibrinogen-like protein 2 M2
1422046_at Itgam integrin alpha M M2
1422122_at Fcer2a Fc receptor, IgE, low affinity II, alpha polypeptide M2
1423141_at Lipa lysosomal acid lipase A M2
1423166_at Cd36 CD36 antigen M2
1423450_a_at Hs3st1 heparan sulfate (glucosamine) 3-O-sulfotransferase 1 M2
1424733_at P2ry14 purinergic receptor P2Y, G-protein coupled, 14 M2
1426172_a_at Cd209a CD209a antigen M2
1426397_at Tgfbr2 transforming growth factor, beta receptor II M2
Attachments
60
Table S1: list of selected M1- and M2-related genes (continued)
Probesets Gene symbol Gene title Polarity
1426642_at Fn1 fibronectin 1 M2
1426807_at Lta4h Leukotriene A4 hydrolase M2
1427683_at Egr2 early growth response 2 M2
1428615_at Lpar6 lysophosphatidic acid receptor 6 M2
1428700_at P2ry13 purinergic receptor P2Y, G-protein coupled 13 M2
1433933_s_at Slco2b1 solute carrier organic anion transporter family, member 2b1 M2
1434034_at Cerk ceramide kinase M2
1438624_x_at Hs3st2 heparan sulfate (glucosamine) 3-O-sulfotransferase 2 M2
1442849_at Lrp1 low density lipoprotein receptor-related protein 1 M2
1446675_at Adk adenosine kinase M2
1448061_at Msr1 macrophage scavenger receptor 1 M2
1448710_at Cxcr4 chemokine (C-X-C motif) receptor 4 M2
1448752_at Car2 carbonic anhydrase 2 M2
1448919_at Cd302 CD302 antigen M2
1448929_at F13a1 coagulation factor XIII, A1 subunit M2
1449015_at Retnla resistin like alpha M2
1449905_at Clec4f C-type lectin domain family 4, member f M2
1449984_at Cxcl2 chemokine (C-X-C motif) ligand 2 M2
1450242_at Tlr5 toll-like receptor 5 M2
1450267_at Tlr8 toll-like receptor 8 M2
1450330_at Il10 interleukin 10 M2
1450430_at Mrc1 mannose receptor, C type 1 M2
1450456_at Il21r interleukin 21 receptor M2
1450488_at Ccl24 chemokine (C-C motif) ligand 24 M2
1450678_at Itgb2 integrin beta 2 M2
1451798_at Il1rn interleukin 1 receptor antagonist M2
1452014_a_at Igf1 insulin-like growth factor 1 M2
1452141_a_at Sepp1 selenoprotein P, plasma, 1 M2
1455876_at Slc4a7 solute carrier family 4, sodium bicarbonate cotransporter, member 7 M2
1456060_at Maf avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene homolog M2
1456250_x_at Tgfbi transforming growth factor, beta induced M2
1457266_at Slc38a6 solute carrier family 38, member 6 M2
1460180_at Hexb hexosaminidase B M2
Att
ach
me
nts
Tab
le S
2: d
iffer
entia
lly e
xpre
ssed
M1-
and
M2-
rela
ted g
enes
in th
e sp
inal
cor
d of
The
iler´
s m
urin
e en
cepha
lom
yelit
is v
irus-
infe
cted
mic
e -
grou
p I:
chem
otax
is
Pro
bese
t G
ene
Sym
bol
Gen
e T
itle
Pol
arity
14
dpi
42
dpi
98
dpi
19
6 dp
i
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
14
195
61_
at
Ccl
3
Che
mo
kine
(C
-C m
otif
) lig
and
3
M1
1
.21
0.1
407
2
.65
0.00
28
2.5
8 0.
0034
2
.80
0.00
20
14
181
26_
at
Ccl
5
Che
mo
kine
(C
-C m
otif
) lig
and
5
M1
8
.89
0.01
18
33
.54
0.00
28
36
.51
0.00
34
17
.61
0.00
20
14
212
28_
at
Ccl
7
Che
mo
kine
(C
-C m
otif
) lig
and
7
M1
1
.01
0.4
537
1
.74
0.00
28
2.1
1 0.
0058
1
.38
0.02
68
14
196
84_
at
Ccl
8
Che
mo
kine
(C
-C m
otif
) lig
and
8
M1
1
.52
0.1
120
4
2.0
9 0.
0028
5
2.8
5 0.
0034
3
0.2
9 0.
0020
14
192
82_
at
Ccl
12
C
hem
oki
ne (
C-C
mo
tif)
liga
nd
12
M1
4
.09
0.01
63
12
.06
0.00
28
12
.93
0.00
34
8.3
3 0.
0020
14
492
77_
at
Ccl
19
C
hem
oki
ne (
C-C
mo
tif)
liga
nd
19
M1
1
.51
0.1
120
2
.45
0.00
28
2.4
0 0.
0107
2
.31
0.00
20
14
234
66_
at
Ccr
7
Che
mo
kine
(C
-C m
otif
) re
cep
tor
7 M
1
1.4
0 0.
0397
1
.43
0.02
63
2.1
3 0.
0425
1
.41
0.02
68
14
186
52_
at
Cxc
l9
Che
mo
kine
(C
-X-C
mo
tif)
liga
nd 9
M
1
6.0
1 0.
0118
1
7.3
1 0.
0028
2
6.8
2 0.
0034
1
4.2
9 0.
0020
14
189
30_
at
Cxc
l10
C
hem
oki
ne (
C-X
-C m
otif
) lig
and
10
M
1
4.2
9 0.
0118
2
0.3
1 0.
0028
2
8.1
5 0.
0034
8
.91
0.00
20
14
196
97_
at
Cxc
l11
C
hem
oki
ne (
C-X
-C m
otif
) lig
and
11
M
1
2.5
1 0.
0397
7
.48
0.00
28
10
.65
0.00
34
4.2
2 0.
0020
14
488
23_
at
Cxc
l12
C
hem
oki
ne (
C-X
-C m
otif
) lig
and
12
M
1
1.0
4 0
.45
37
1.3
1 0.
0384
1
.14
0.1
034
2
.02
0.00
20
14
487
10_
at
Cxc
r4
Che
mo
kine
(C
-X-C
mo
tif)
rece
pto
r 4
M
2
1.1
4 0
.32
64
2.5
5 0.
0028
3
.86
0.00
34
5.5
2 0.
0020
14
188
26_
at
Ms4
a6
b
Me
mb
rane
-sp
ann
ing
4-d
om
ains
, su
bfa
mily
A,
me
mb
er 6
B
M2
3
.42
0.01
63
9.0
1 0.
0028
1
2.4
4 0.
0034
9
.86
0.00
20
14
203
80_
at
Ccl
2
Che
mo
kine
(C
-C m
otif
) lig
and
2
M2
1
.11
0.1
729
2
.31
0.00
28
2.3
6 0.
0034
1
.47
0.00
20
14
178
51_
at
Cxc
l13
C
hem
oki
ne (
C-X
-C m
otif
) lig
and
13
M
1,
M2
2
3.6
6 0.
0118
9
6.9
8 0.
0028
1
28
.49
0.00
34
59
.02
0.00
20
dp
i = d
ays
po
st in
fect
ion;
FC
= fo
ld c
han
ge; b
old
q-v
alue
s d
isp
lay
sig
nific
ant
diff
ere
nce
s b
etw
ee
n in
fec
ted
mic
e a
nd m
ock
-in
fect
ed c
ont
rol m
ice
61
Att
ach
me
nts
Tab
le S
2 (c
ontin
ued)
: diff
eren
tially
exp
ress
ed M
1- an
d M
2-re
late
d ge
nes
in th
e sp
inal
cor
d of
The
ilers
mur
ine
ence
phal
omye
litis
viru
s-in
fect
ed m
ice
- gr
oup
II (p
hago
cyto
sis,
ant
igen
proc
essi
ng a
nd p
rese
ntat
ion
Pro
bese
t G
ene
Sym
bol
Gen
e T
itle
M1/
M2
Mar
ker
14 d
pi
42 d
pi
98 d
pi
196
dpi
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
14
178
76_
at
Fcg
r1
Fc
rece
pto
r, I
gG,
high
affi
nity
I
M1
1
.95
0.01
18
5.0
4 0.
0028
5
.81
0.00
34
3.9
4 0.
0020
14
354
77_
s_at
F
cgr2
b
Fc
rece
pto
r, I
gG,
low
affi
nity
II
b M
1
1.7
5 0.
0118
8
.52
0.00
28
15
.02
0.00
34
12
.12
0.00
20
14
486
20_
at
Fcg
r3
Fc
rece
pto
r, I
gG,
low
affi
nity
III
M
1
1.6
2 0.
0118
2
.98
0.00
28
4.3
1 0.
0034
3
.69
0.00
20
14
171
89_
at
Psm
e2
Pro
teas
om
e (p
roso
me
, m
acr
op
ain)
28
su
bu
nit,
bet
a
M1
1
.35
0.01
63
2.2
0 0.
0028
2
.65
0.00
34
2.1
7 0.
0020
14
506
96_
at
Psm
b9
P
rote
aso
me
(pro
som
e,
mac
rop
ain
) su
bu
nit,
be
ta t
ype
9 (
larg
e
mu
ltifu
nctio
nal p
eptid
ase
2)
M1
3
.88
0.01
18
11
.92
0.00
28
16
.42
0.00
34
12
.33
0.00
20
14
539
13_
a_at
T
ap2
Tra
nsp
ort
er 2
, A
TP
-bin
din
g ca
sse
tte,
sub
-fa
mily
B (
MD
R/T
AP
) M
1
1.6
2 0.
0118
2
.80
0.00
28
3.3
7 0.
0034
2
.53
0.00
20
14
218
12_
at
Tap
bp
TA
P b
ind
ing
pro
tein
M
1
1.4
1 0.
0163
2
.55
0.00
28
3.5
6 0.
0034
2
.70
0.00
20
14
392
21_
s_at
C
d4
0
CD
40
ant
igen
M
1
1.1
6 0
.25
17
2.0
7 0.0
028
2.0
7 0.
0034
1
.39
0.00
68
14
183
40_
at
Fce
r1g
Fc
rece
pto
r, I
gE,
high
affi
nity
I,
gam
ma
po
lyp
ep
tide
M
2
1.8
3 0.
0118
4
.38
0.00
28
5.1
0 0.
0034
4
.39
0.00
20
14
218
55_
at
Fgl
2
Fib
rino
gen
-like
pro
tein
2
M2
1
.69
0.1
729
4
.90
0.00
28
6.2
2 0.
0034
4
.86
0.00
20
14
504
30_
at
Mrc
1
Ma
nno
se r
ece
pto
r, C
typ
e 1
M
2
1.0
7
0.4
537
1
.33
0.0
263
1
.73
0.01
07
2.3
8 0.
0020
14
480
61_
at
Msr
1
Mac
rop
hage
sca
ven
ger
rece
pto
r 1
M2
1.0
7 0
.45
37
2.6
6 0.
0028
4
.74
0.00
34
4.2
2 0.
0020
14
524
31_
s_at
H
2-A
a
His
toco
mp
atib
ility
2,
clas
s II
ant
ige
n A
, al
pha
M
1,
M2
5
.49
0.01
18
29
.30
0.00
28
37
.55
0.00
34
29
.10
0.00
20
14
206
99_
at
Cle
c7a
C-t
ype
lect
in d
om
ain
fam
ily 7
, m
em
be
r a
M
2
1.3
9 0
.21
25
8.2
0 0.
0028
1
5.8
6 0.
0034
1
3.0
8 0.
0020
14
498
58_
at
Cd
86
C
D8
6 a
ntig
en
M1
, M
2
1.4
6 0.
0118
4
.42
0.00
28
5.6
5 0.
0034
3
.58
0.00
20
14
175
97_
at
Cd
28
C
D2
8 a
ntig
en
M2
1
.08
0.3
264
1
.91
0.00
28
3.2
0 0.
0058
2
.70
0.00
20
dp
i = d
ays
po
st in
fect
ion;
FC
= fo
ld c
han
ge; b
old
q-v
alue
s d
isp
lay
sig
nific
ant
diff
ere
nce
s b
etw
ee
n in
fec
ted
mic
e a
nd m
ock
-in
fect
ed c
ont
rol m
ice
62
Att
ach
me
nts
Tab
le 2
(co
ntin
ued)
: diff
eren
tially
exp
ress
ed M
1- an
d M
2-re
late
d ge
nes
in th
e sp
inal
cor
d of
The
iler´
s mur
ine
ence
phal
omye
litis
viru
s-in
fect
ed m
ice
- gr
oup
III: c
ytok
ine
sign
allin
g an
d gr
owth
fact
ors
Pro
be s
et
Gen
e S
ymbo
l G
ene
Titl
e M
1/M
2 M
arke
r 14
dpi
42
dpi
98
dpi
19
6 dp
i
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
14
490
25_
at
Ifit3
In
terf
ero
n-i
nduc
ed p
rote
in w
ith
tetr
atri
cop
ep
tide
rep
eats
3
M1
3
.65
0.01
18
10
.87
0.00
28
11
.96
0.00
34
5.2
3 0.
0020
14
259
47_
at
Ifng
In
terf
ero
n ga
mm
a
M1
1
.16
0.2
517
2
.02
0.00
28
2.6
9 0.
0034
1
.59
0.04
03
14
485
75_
at
Il7r
Inte
rleu
kin
7 r
ece
pto
r M
1
1.2
2 0
.14
07
2.0
1 0.
0028
3
.42
0.00
34
3.8
5 0.
0020
14
484
36_
a_
at
Irf1
In
terf
ero
n re
gul
ato
ry f
act
or
1
M1
1
.51
0.02
82
3.4
7 0.
0028
4
.40
0.00
34
3.1
1 0.
0020
14
172
44_
a_
at
Irf7
In
terf
ero
n re
gul
ato
ry f
act
or
7
M1
2
.66
0.01
63
10
.12
0.00
28
11
.42
0.00
34
4.4
6 0.
0020
14
243
39_
at
Oa
sl1
2
'-5' o
ligo
ade
nyl
ate
syn
the
tase
-like
1
M
1
1.6
1 0.
0163
2
.81
0.00
28
3.6
5 0.
0034
1
.99
0.00
20
14
250
65_
at
Oas
2
2'-5
' olig
oa
den
yla
te s
ynth
eta
se 2
M
1
1.3
2 0.
0282
2
.26
0.00
28
2.5
9 0.
0034
2
.08
0.00
20
14
206
53_
at
Tgf
b1
T
rans
form
ing
gro
wth
fa
cto
r, b
eta
1
M2
1
.16
0.21
25
1.6
1 0.
0028
2
.13
0.00
34
1.9
8 0.
0020
14
562
50_
x_a
t T
gfb
i T
rans
form
ing
gro
wth
fa
cto
r, b
eta
in
duc
ed
M
2
1.0
9 0.
3743
2
.04
0.00
51
2.3
3 0.
0034
2
.09
0.00
20
14
263
97_
at
Tgf
br2
T
rans
form
ing
gro
wth
fa
cto
r, b
eta
re
cep
tor
II M
2
1.3
9 0.
0118
1
.91
0.00
28
1.9
9 0.
0034
2
.11
0.00
20
14
520
14_
a_
at
Igf1
In
sulin
-lik
e gr
ow
th f
acto
r 1
M
2
1.0
7 0.
4858
1
.95
0.00
28
4.4
5 0.
0034
4
.99
0.00
20
14
517
98_
at
Il1rn
In
terl
euki
n 1
re
cep
tor
ant
ago
nis
t M
2
1.2
2 0.
1120
2
.00
0.00
28
2.3
8 0.
0058
1
.59
0.00
20
dp
i = d
ays
po
st in
fect
ion;
FC
= fo
ld c
han
ge; b
old
q-v
alue
s d
isp
lay
sig
nific
ant
diff
ere
nce
s b
etw
ee
n in
fec
ted
mic
e a
nd m
ock
-in
fect
ed c
ont
rol m
ice
Tab
le 2
(co
ntin
ued)
: diff
eren
tially
exp
ress
ed M
1- an
d M
2-re
late
d ge
nes
in th
e sp
inal
cor
d of
The
iler´
s mur
ine
ence
phal
omye
litis
viru
s-in
fect
ed m
ice
- gr
oup
IV: T
oll-l
ike
rece
ptor
sig
nallin
g
Pro
be s
et
Gen
e S
ymbo
l G
ene
Titl
e M
1/M
2 M
arke
r 14
dpi
42
dpi
98
dpi
19
6 dp
i
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
14
490
49_
at
Tlr1
T
oll-
like
re
cep
tor
1 M
1
1.2
5 0
.17
29 2
.29
0.00
28
2.8
1 0.
0034
2
.51
0.00
20
14
191
32_
at
Tlr2
T
oll-
like
re
cep
tor
2 M
1,
M2
1
.77
0.01
63
2.6
6 0.
0028
3
.31
0.00
34
2.9
4 0.
0020
dp
i = d
ays
po
st in
fect
ion;
FC
= fo
ld c
han
ge; b
old
q-v
alue
s d
isp
lay
sig
nific
ant
diff
ere
nce
s b
etw
ee
n in
fec
ted
mic
e a
nd m
ock
-in
fect
ed c
ont
rol m
ice
63
Att
ach
me
nts
Tab
le 2
(co
ntin
ued)
: diff
eren
tially
exp
ress
ed M
1- an
d M
2-re
late
d ge
nes
in th
e sp
inal
cor
d of
The
iler´
s mur
ine
ence
phal
omye
litis
viru
s-in
fect
ed m
ice
- gr
oup
V: a
popt
osis
-rel
ated
gen
es
Pro
be s
et
Gen
e S
ymbo
l G
ene
Titl
e M
1/M
2 M
arke
r 14
dpi
42
dpi
98
dpi
19
6 dp
i F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e
14
190
04_
s_at
B
cl2
a1a
B-c
ell l
euke
mia
/lym
pho
ma
2 r
ela
ted
p
rote
in A
1a
/// B
-cel
l le
uke
mia
/lym
pho
ma
2 r
elat
ed
pro
tein
A1
b //
/ B-c
ell
leuk
em
ia/ly
mp
hom
a 2
rel
ated
p
rote
in A
1d
M1
1
.82
0.01
18
5.7
7 0.
0028
8
.40
0.00
34
6.0
6 0.
0020
14
213
92_
a_
at
Bir
c3
Ba
culo
vira
l IA
P r
epe
at-c
ont
aini
ng
3
M1
1
.29
0.03
97
1.9
9 0.
0028
2
.01
0.00
34
2.0
0 0.
0020
14
513
18_
a_
at
Lyn
Y
am
agu
chi s
arco
ma
vira
l (v-
yes-
1)
onc
oge
ne h
om
olo
g
M1
1
.44
0.01
18
2.3
7 0.
0028
2
.64
0.00
34
2.3
1 0.
0020
14
163
82_
at
Cts
c C
ath
epsi
n C
M
2
1.8
6 0.
0163
4
.84
0.00
28
6.8
0 0.
0034
5
.57
0.00
20
dp
i = d
ays
po
st in
fect
ion;
FC
= fo
ld c
han
ge; b
old
q-v
alue
s d
isp
lay
sig
nific
ant
diff
ere
nce
s b
etw
ee
n in
fec
ted
mic
e a
nd m
ock
-in
fect
ed c
ont
rol m
ice
Tab
le 2
(co
ntin
ued)
: diff
eren
tially
exp
ress
ed M
1- an
d M
2-re
late
d ge
nes
in th
e sp
inal
cor
d of
The
iler´
s mur
ine
ence
phal
omye
litis
viru
s-in
fect
ed m
ice
- gr
oup
VI:
extr
acel
lula
r m
atrix
rec
epto
r in
tera
ctio
n an
d ce
ll ad
hesi
on m
olec
ules
Pro
be s
et
Gen
e S
ymbo
l G
ene
Titl
e M
1/M
2 M
arke
r 14
dpi
42
dpi
98
dpi
19
6 dp
i F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e 4
24
067
_at
Ic
am
1
Inte
rcel
lula
r ad
hesi
on
mo
lecu
le 1
M
1
1.6
7 0.
0397
3
.90
0.00
28
5.9
0 0.
0034
4
.08
0.00
20
14
237
60_
at
Cd
44
C
D4
4 a
ntig
en
M
1
1.1
6 0
.41
32
2.3
0 0.
0028
2
.29
0.00
34
2.6
9 0.
0020
14
231
66_
at
Cd
36
C
D3
6 a
ntig
en
M
2
1.1
1 0
.29
29
1.3
6 0.
0559
3
.95
0.00
34
3.2
0 0.
0020
14
506
78_
at
Itgb
2
Inte
grin
bet
a 2
M
2
1.9
1 0.
0118
5
.18
0.00
28
7.4
8 0.
0034
6
.66
0.00
20
14
220
46_
at
Itga
m
Inte
grin
alp
ha M
M
2
1.1
7 0
.14
07
2.
04
0.00
28
2.4
3 0.
0034
2
.20
0.00
20
dp
i = d
ays
po
st in
fect
ion;
FC
= fo
ld c
han
ge; b
old
q-v
alue
s d
isp
lay
sig
nific
ant
diff
ere
nce
s b
etw
ee
n in
fec
ted
mic
e a
nd m
ock
-in
fect
ed c
ont
rol m
ice
64
Att
ach
me
nts
Tab
le 2
(co
ntin
ued)
: diff
eren
tially
exp
ress
ed M
1- an
d M
2-re
late
d ge
nes
in th
e sp
inal
cor
d of
The
iler´
s mur
ine
ence
phal
omye
litis
viru
s-in
fect
ed m
ice
- gr
oup
VII:
mis
cella
neou
s ge
nes
Pro
bese
t G
ene
Sym
bol
Gen
e T
itle
M1/
M2
Mar
ker
14 d
pi
42 d
pi
98 d
pi
196
dpi
FC
q-
valu
e F
C
q-va
lue
FC
q-
valu
e F
C
q-va
lue
14
493
63_
at
Atf
3
Act
ivat
ing
tran
scri
ptio
n fa
cto
r 3
M1
1.0
6 0
.45
37
1.6
5 0.
0028
2
.33
0.00
34
2.0
9 0.
0038
14
195
49_
at
Arg
1
Arg
ina
se
M2
-1
.09
0
.41
32
4.5
2 0.
0028
9
.99
0.00
34
8.0
9 0.
0020
14
189
82_
at
Ce
bp
a
CC
AA
T/e
nha
nce
r b
ind
ing
pro
tein
(C
/EB
P),
alp
ha
M2
1
.16
0.0
862
1
.44
0.00
28
2.0
8 0.
0034
2
.17
0.00
20
14
197
64_
at
Chi
3l3
C
hitin
ase
3-li
ke 3
M
2
-1.0
0
0.5
432
1
.07
0.4
307
1
.82
0.1
284
3
.47
0.00
68
14
601
80_
at
Hex
b
He
xosa
min
ida
se B
M
2
1.3
4 0.
0163
1
.96
0.00
28
2.3
9 0.
0034
2
.24
0.00
20
65
Acknowledgments
66
10. ACKNOWLEDGMENTS
I would never have been able to finish my dissertation without the guidance of my supervisors,
colleagues and my family. The contributions of many kind people, in their different ways, have made
this possible. I take this opportunity to express
my special appreciation and thanks to Prof. Wofgang Baumgärtner, he has been a tremendous
mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as
a research scientist. His support on both research as well as on my career have been priceless. The
countless his times supervise me during my study will not be forgotten and not express enough
thanks to him.
my deepest gratitude to Prof. Andreas Beineke for his excellent guidance, caring, patience,
enthusiastic encouragement, useful critiques and providing me with an excellent atmosphere for
doing research. Thanks also for his advice, assistance in keeping my progress on schedule and
patiently corrected my writing. My grateful thanks are also extended for helping me to develop my
background in virology and pathology. Last but not least, special thanks goes to for him support and
encouragement throughout my study.
words which are not enough to thank to Dr. Vanessa Herder, who as my supervisor also my
colleague also my best friend. I have been inspired by her meticulousness, attention to detail and
energetic application to any problem. When I stay in the same room with her, it is truly a wonderful
place to study and work. Her involvements into my research and concern for my welfare have greatly
motivated me. With great pleasure, I thank to for her help to make things available. Her support,
guidance, advice are greatly appreciated. Indeed, without his guidance, I would not be able to put
many things together. Thanks Vanessa.
a big sense of gratitude to Prof. Martin Stangel, Prof. Andrea Tipold and Dr. Reiner Ulrich for his/her
cordial support, valuable information and guidance, which helped me in completing this study
through various stages.
special thanks to Florian, Max, Annika and Johannes who supported me for sustained and incited me
to strive towards my goal. Thanks guys for the enjoyable moment as well as brilliant comments and
suggestions.
the grateful for cooperation during the period of my study which provided by Margo, Akram, Kristel,
Nadeem, Barbara, Ning, Armend, Eva, Witchaya, Rike, and Adnan. All of them have been there to
support me when I recruited task during my study. Best wishes and many thanks for them.
my thanks to Caro, Kerstin, Petra, Bettina, Angelika, Leila, Danuta, Claudia, Frau Bährens, Herr
Kuhlmann and Herr Jewgenow for their guidance and motivation.
thank you to DAAD/ACEH scholarship excellence, ZSN and DFG for allowing me to take the financial
opportunity. My completion of this project could not have been accomplished without the support.
great pleasure to the Pathology Department of Veterinary Medicine University Hannover-Germany,
Center of Neuroscience Hannover-Germany as well as Veterinary Medicine Faculty of Syiah Kuala
University Aceh-Indonesia for the opportunity and support to me.
sincere gratitude to Agus, Lisa, Shahril, Zafran, Falah, Indera, Hilda, Nana, Jahar, France, Sandro,
Ismi, Hamdan, Izza, Haqi and Ernah for the supporting and memorable time of our colorful and
happy during my study in Germany.
appreciation and grateful to my beloved husband, son and daughters. Words cannot express how
grateful also to my parents, my mother in law and my sisters. Their patience and understanding,
which is support me in all of moments during my study. Their love will be the answer of my queries
when there is nobody able to solve it.
At the end, I wish to thank everybody who helps me but I am not mention here.
ISBN 978-3-86345-232-2
Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375
E-Mail: [email protected] · Internet: www.dvg.de
Polarization ofimmune cells inTheiler‘s murineencephalomyelitis
Cut Dahlia Iskandar
Hannover 2014
Department of Pathology. Centre for Systems Neuroscience.University of Veterinary Medicine.